Reviews in Medical Virology

Rev. Med. Virol. 8: 213222 (1998)

Virus Assembly and

Disassembly: the Adenovirus
Cysteine Protease as a Trigger
Factor
Urs F. Greber
Institute of Zoology, University of Zrich, Winterthurerstrasse 190, CH-8057 Zrich, Switzerland
SUMMARY
Viruses are efficient carriers of genetic material between cells. They specifically recognise a
target cell and utilise host functions for genome delivery to the replication site. A mature viral
capsid emerging from an infected cell serves at least three distinct functions. It enables virus
egress from the infected cell, protects the extracellular genome against chemical and physical
stress and mediates virus entry into a non-infected cell. How can a virus particle be stably
assembled in an infected cell and moments laterafter passing through the extracellular
milieube disintegrated by a non-infected cell? In this review I discuss how adenovirus, a
DNA virus, recruits cellular and viral factors and makes use of its own cysteine protease to
regulate capsid assembly and disassembly.  1998 John Wiley & Sons, Ltd.
Accepted 12 May 1998

INTRODUCTION
Virus capsids are constructed in a way to allow firm
packaging of the genome and also efficient unpacking and
timely genome activation inside a host cell. Capsids vary
in size from dozens to hundreds of nanometers and
surround genomes which can be a few thousands up to
several hundred thousands of nucleotides long. With the
discovery of the genetic code it was recognised that any
given viral genome was too small to encode a single
polypeptide making up the entire capsid. It was found
instead, that a capsid was constructed of one or several
smaller polypeptides arranged as symmetric building
elements.1 Viral capsids occur in either helical or icosahedral symmetry. Helical symmetry is used, e.g. by the
tobacco mosaic virus, whose coat protein makes identical
contacts to all its neighbors, except for the subunits at
both ends of the helix.2 Icosahedral shells can be built by
multiples of 60 subunits provided that the subunit
contacts are variable depending on the position in the
icosahedron.3 Different contacts between subunits can
arise by conformational changes induced during the
cooperative capsid assembly process.4,5 Cooperative
assembly of capsids takes place in an infected cell at high
concentrations of free subunits and can occur also in cell
free systems.
Abbreviations used: ad2, adenovirus type 2; ADP, adenovirus death
protein; CAR, coxsackie virus, adenovirus receptor; DTT, dithiothreitol; NEM, N-ethyl-maleimide; ND10, nuclear domain 10; NPC, nuclear
pore complex; PTP, preterminal protein; NLS, nuclear localisation
signal; SV40, simian virus 40.
CCC 10529276/98/04021310 $17.50
 1998 John Wiley & Sons, Ltd.

Control of capsid assembly may be exerted either at

the level of capsid formation or of genome packaging.
Capsid formation may be assisted by scaffolding structures or cellular chaperones, both of which are not parts
of the final capsid structure.4,6 Packaging of the nucleic
acid can occur by at least three different mechanisms.
(I) A proteinaceous capsid is built around a condensed
nucleic acid, (II) a capsid shell is built and the nucleic acid
packed and condensed within the shell, (III) the nucleic
acid is condensed sequentially within the growing capsid.
All three strategies and variations thereof are used by
different viruses.7
Directionality of assembly can also be conferred by
limited proteolysis of capsid proteins.810 These reactions
are catalysed by either host or virally encoded enzymes
and may stabilise or destabilise the capsid depending on
the virus.11 In the case of enveloped viruses, proteolytic
processing of surface spike proteins also confers
additional functions, such as the ability to fuse membranes.12 Proteolytic maturation can either remove protein fragments, which are not needed in the mature
virion, or switch the function of a particular capsid
protein from an assembly to a disassembly effector.
Besides proteolytic modifications, virus capsids can be
altered posttranslationally by the formation of intra- or
intermolecular disulphide bonds as shown for enveloped
viruses13 and non-enveloped viruses.14
Conceptually, capsid disassembly is the transition from
an ordered structure to a disordered structure with
several intermediates. This reaction may occur as a
sequence of endothermic reactions each leading to a new

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U. F. GREBER

Figure 1. The assembly/disassembly paradox in virus exit and entry (for explanations, see text).

local energy minimum until a global minimum of the

disassembled particle is reached. In the case of the
Salmonella phage P22, differential scanning calorimetry
combined with different biochemical techniques has
measured such energetic transitions during the expansion
of the procapsids.15 Four distinct thermal intermediates
were observed, endothermic reactions at 49C, 71C and
87C and one exothermic reaction at 61C. The endothermic reactions corresponded to denaturation (and exit)
of the internal scaffolding protein, to denaturation of
individual capsid proteins and finally to dissociation of
the capsid. The exothermic reaction coincided with
expansion of the procapsid and was inhibited by a kinetic
barrier. It is expected that cellular factors act to decrease
not only this barrier, but also participate in catalysing the
major endothermic transitions.
Determining common biophysical parameters of disassembly and assembly paths may thus give insights into
the missing molecular mechanisms. Such approaches can
relate to emerging cell biological questions including the
nature of the trigger factors which initiate assembly and
disassembly programmes. Solving such questions will
help to understand how a virus particle can be stably
assembled in an infected cell and moments laterafter
passing through the extracellular milieucan be disassembled.
Four principal ideas can explain such an apparent
paradox (see also Figure 1):

inhibited by direct or indirect inactivation of ribosomes in

the infected cell, which may involve newly synthesised
capsid protein.19,20
3. The path of entry is different from the path of egress.
The incoming virus either interacts with different host
factors or signals the initiation of host responses which
are different from those of the outgoing virus. Many
enveloped viruses, which bud from either internal membranes or from the plasma membrane, are released in
chronic or persistent infections long before the host cell
dies.2123 For most non-enveloped viruses, specific
release mechanisms have long been suspected for exit,
but are so far poorly characterised. Interesting exceptions
are the polarised release of poliovirus and simian virus 40
(SV40).2426
4. Virus release occurs by cell lysis after cell death as a
general mechanism for setting free large amounts of
virions. Such mechanisms operate at late stages of many
virus infections.

1. The incoming virion is different from the outgoing

virion. This is probably true for most viruses owing to
the stepwise assembly and disassembly programmes.
Enveloped viruses shed their lipid and spike proteins at
the plasma membrane or at an endosomal membrane and
then release a capsid-associated genome into the cytoplasm, while non-enveloped viruses interact directly with
cell surface receptors.1618
2. Cellular or viral factors involved in virus entry or
egress are either activated or inactivated during infection.
For example, alpha virus capsid uncoating may be

Human adenoviruses naturally infect epithelial cells of the

upper and lower respiratory tract, the gastrointestinal or
urinary tracts and rarely cause keratoconjunctivitis in the
visual system.27 They are classified into subgroups A to
F. The subgroup C comprises ad2 and its closely related
serotype ad5. These viruses enter human airway cells of
the upper respiratory tract and also lymphoid tissue
causing latent throat infections which can sporadically
emerge as respiratory illnesses. Like all other adenoviruses, ad2 produces progeny virions within the nucleus of
the infected cell. When rendered replication defective,

 1998 John Wiley & Sons, Ltd.

Unlike other viruses, adenovirus appears to regulate its

assembly and disassembly processes by avoiding
untimely activation of a single enzyme, its own cysteine
protease p23. In this respect, it belongs to category 2, but
its assembly and disassembly pathways are distinct in
many other respects (see below).

ADENOVIRUS TYPE 2 PARTICLE (AD2)

Rev. Med. Virol. 8: 213222 (1998)

ASSEMBLY AND DISASSEMBLY

215

double-stranded genomic DNA in bacteria, such as E. coli.

Also inside the adenovirus capsid are 10 to 50 copies of
the cysteine protease p23.3537 p23 has most likely no
major role in packaging or condensing the genome, since
a mutant ad2 ts1, which contains five times less protease
than wild type virions, packs genomes of wild type
length into normally sized capsids.

ASSEMBLY

Figure 2. Schematic view of the adenovirus type 2 particle.

ad2 and ad5 are broadly used as gene transfer vehicles for
somatic cell therapy.28
The adenovirus particle is of icosahedral symmetry and
has a diameter of approximately 90 nm. Many of its
polypeptides, of which there are at least 11 different ones,
have been localised by cryo-electron microscopy and
3-dimensional image reconstruction depicted in Figure 2
and extensively reviewed in.29 The particle shell is built
from 252 capsomers, 240 of which are hexon capsomers
building the facets. Each hexon is a trimer with a
hexagonal base facing the inside and a pyramidal top to
the outside of the capsid. A number of minor capsid
proteins are inserted into the icosahedron at specific
positions. Protein IIIa links adjacent facets by spanning
the capsid. Protein IX stabilises groups of nine trimeric
hexons, and protein VI anchors the ring of peripentonal
hexons by bridging to the DNA core inside the capsid.
Other minor proteins, such as protein VIII, X, XI or XII
have not been localised unequivocally but are likely to
occur at the inside of the capsid. The vertices of the
icosahedron are made up by 12 penton base capsomers,
each arranged in a ring-like configuration with a central
cavity. Inserted into each cavity is the trimeric fibre
protein consisting of a linear shaft and a carboxy-terminal
globular head domain pointing to the outside.
Inside the capsid is a single copy of a linear doublestranded DNA molecule of about 36 kbp. The DNA is
condensed with proteins V, VII and the minor component
and covalently linked to two terminal proteins sitting
at each a 5 end.30 It is not known exactly whether the
DNA is organised into nucleosome-like structures or is
packaged into crystalline or liquid crystalline arrays as in
Loligo sperm heads, bacteriophages or herpes simplex
virus.3134 Based on an estimated inner capsid diameter
of about 70 nm, the adenovirus genome is packaged at a
density similar to double-stranded DNA in crystalline
form.5 This would require an estimated 6000 fold condensation compared with the minimally condensed
 1998 John Wiley & Sons, Ltd.

A detailed picture of the adenovirus assembly pathway

has been difficult to obtain due to incomplete knowledge of the proteins involved, the lability of assembly
intermediates and the lack of an in vitro system.
Similar to the building of another icosahedral capsid,
the human herpes simplex virus capsid,38 the development of temperature-sensitive assembly mutants and the
introduction of gentle isolation procedures together with
an increasingly detailed picture of the capsid structure
emerging over the past 15 to 20 years have enabled a
broad picture of the assembly events to be drawn
(depicted in Figure 3 and summarised in39,40).
The first step to assembly is the formation of hexon,
penton base and fibre capsomers. While fibre and penton
base oligomerisations occur independently of chaperones,
hexon trimers are formed in the cytoplasm by a transient,
direct association of nascent polypeptide with a chaperone of Mr 100 000 in a 1:1 complex.41 This assisted
folding pathway is thought to help to avoid assembly
errors. A completely folded hexon trimer is then
imported into the nucleus perhaps in association with the
precursor of protein VI.42
Typical assembly intermediates can be generated from
cells infected with temperature-sensitive mutants by
pulse labelling experiments with radioactive amino acids
and orthophosphate.43,44 Again, the formation of subcomplexes is thought to help minimise assembly errors.
These experiments suggested that assembly proceeds
through the formation of empty capsids. Empty capsids
have a characteristic buoyant density of 131 g/mL
and are made up of hexon, penton base, fibre, and precursors of proteins IIIa, VI and VIII, but lack DNA. Empty
capsids also contain scaffolding proteins L1 52/55K45 and
perhaps minor proteins of unknown function.46
The next intermediate which can be readily isolated is
the so called heavy intermediate of 1.37 g/mL. Besides
the proteins found in empty capsids, it contains DNA
covalently linked to the precursor of the terminal protein,
but lacks most of the scaffolding proteins and the core
proteins V, VII and . It is likely that the core proteins
have been lost during the isolation procedure, since
newly replicated viral DNA in the nucleus late in
infection is associated with proteins V and VII.40 The
down-regulation of histone synthesis late in infection
may be the reason for the preferred packaging of viral
DNA associated with core proteins rather than histones.
How the DNA is forced into the procapsid is currently
unknown. Evidence both in favour and against
replication-coupled packaging of DNA can be found in
the literature.40 It is clear, however, that gene products of
Rev. Med. Virol. 8: 213222 (1998)

216

U. F. GREBER

Figure 3. The adenovirus replication cycle (for explanations, see text).

the late region 1 (L1), such as the scaffolding proteins

of Mr 52 000 and 55 000, respectively, and the precursor
of protein IIIa, are involved in DNA encapsidation, since
ts mutants in either of these proteins accumulate light
assembly intermediates.44,45 It is also clear that packaging
somehow depends on a cis-acting DNA sequence containing AT-rich repeats at the left end of the viral
genome.47 Possibly, specific trans-acting packaging factors recognise this and adjacent DNA regions and thus
facilitate polar encapsidation of viral DNA into empty
capsids.48 Whether specific proteins are added to the
capsid once it is filled with DNA in order to lock-in the
DNA is unknown.
The subsequent assembly intermediate is represented
by the so called young virions.49 They are most
prominent in the ts1 mutant at the restrictive temperature
and arise due to the lack of functional protease p23.35,50
Young virions have the same specific density as wild
type particles (134 g/mL), but lack processing of precursor polypeptides pIIIa, pVI, pVII, pVIII, p and preterminal protein (pTP) and are devoid of polypeptides X, XI
and XII. The latter polypeptides are perhaps proteolytic
processing products from precursor proteins. Young
virions appear to contain the Mr 55 000 scaffolding
protein.51 The vertex region of young virions has not
been processed and so is structurally different from the
vertex of mature capsids.52 This could account for the failure of ad2 ts1 to penetrate endosomal membranes.5355
The mutation in ts1 responsible for the lack of processing has been mapped to a C-T transition resulting in a
proline to leucine exchange at position 137 of the p23
proteinase.56
 1998 John Wiley & Sons, Ltd.

CYSTEINE PROTEASE P23

p23 is found in wild type virions, but not in ts1
particles.57,58 It is encoded in the genomic region L3 and
responsible for the conversion of the procapsid to a
mature, infectious particle.14,36,59 p23 is a cysteine protease of 204 amino acids containing eight cysteine
residues.60 Enzyme activity can be completely abolished
with low concentrations of reducing agents and an excess
of alkylating agents, such as iodoacetamide or N-ethylmaleimide (NEM).61 Extensive mutagenesis experiments
have demonstrated that cysteines at positions 104 and
122 are critical for hydrolytic activity.62 Biochemical
inhibitor studies, further mutagenesis experiments and
the crystal structure of the enzyme indicate that the
catalytic mechanism utilises a catalytic triad, Cys122His54-Glu71, similar to papain.6365 Two alternative
consensus cleavage motif for p23 have been identified
through analysis of cleaved precursor proteins during
virus infection and the use of synthetic peptides as
substrates: M(I or L)XGX-G or M(I or L)XGG-X (with the
scissile bond indicated as -).66
p23 activity of purified wild type virus or from
recombinant bacteria can be stimulated by addition of
lysates from ts1 infected cells suggesting that cofactors
are needed for maximal activity.67 The first cofactor
identified has been the 11 amino acid C-terminal peptide
of the precursor to protein VI, pVI.14 This activating
peptide contains a critical cysteine residue at the
C-terminal position -2, which engages in a disulphide
exchange reaction with cysteine 104 of p23 as indicated
by biochemical studies and the crystal structure of p23
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ASSEMBLY AND DISASSEMBLY

bound to the activator peptide.64,68 Possibly, a second

activator of p23, polyanions, including DNA, either
stimulate p23 activity or stabilise the enzyme.37
Activity of p23 from wild type virions is enhanced in
the presence of reducing agents suggesting that within
the virion the enzyme is inactive, perhaps disulphidebonded.14 Peptide-activated recombinant protease is,
however, inhibited by low concentrations of reducing
agent suggesting that reducing agents inhibit the
activation process but stimulate the enzymatic activity.
Given the fact that adenovirus assembly in all its
complexity strongly depends on the p23 protease it is
possible that capsid proteolysis is temporally controlled.
The minimal requirement for processing is capsid assembly itself as indicated by assembly-defective mutants.40
An additional requirement for processing is that protease
p23 is properly packaged into nascent virions. Owing to
its basic isoelectric point, p23 co-purifies with viral cores
and associates nonspecifically with DNA.59 It is possible
that p23 enters the capsid, perhaps in association with the
viral DNA as a complex with proteins V and VII. Once
inside the capsid, correctly folded wild type p23, but not
mutant ts1 p23, could interact with pVI.69 This interaction would directly or indirectly lead to N- and
C-terminal cleavage of pVI and the production of the
activating peptide. Interestingly, the activating peptide of
pVI contains four basic amino acids (R and K) which
might serve as a binding determinant of pVI but not of
the processed protein VI to DNA core. This could be part
of a conformational change enabling the fully processed
pVI to stably lock-in with hexon. In agreement with such
a scenario, blot overlay experiments have detected an
interaction between hexon and processed pVI, but not
with the precursor of VI.70,71 p23 would now engage in
processing all the capsid proteins with available consensus sequences including the scaffolding proteins until a
mature virion emerges. Proteolytic activity would be
both self-limiting and diminished as soon as the virions
encounter oxidising conditions, e.g. in the extracellular
medium. How the scaffolding proteins exit the capsid is
unknown. It is possible that they leave through openings
in an incomplete capsid, or via transient holes created
by conformational changes in the shell, analogous to
bacteriophage maturation.72

RELEASE
The mechanisms of adenovirus release from the infected
cell are largely unknown.30 While small enveloped
viruses, such as retroviruses, can be non-lytic and shed
virus particles over the entire life time of the host,
non-enveloped viruses, such as adenoviruses, polio
viruses or papovaviruses are released from cells in large
amounts late in infection due to cell lysis. Lytic release of
adenovirus is facilitated by a virally encoded Mr 11 600
polypeptide also called ADP (adenovirus death protein)
of the delayed early transcription unit E3.73,74 E3-11.6K
mutants replicate and assemble capsids with apparently
normal efficiencies, but fail to lyse their hosts up to about
 1998 John Wiley & Sons, Ltd.

217

5 days post infection. As early as 1 day post infection,

ADP mutant virions appear in the extracellular medium at
low levels, about 4 logs lower in abundance than the
intracellular virus load. All the released particles are
proteolytically processed suggesting that capsid processing is essential for release. Interestingly, unprocessed ts1
mutant particles form much smaller plaques than wild
type virus suggestingbut not provingthat ts1 particles are impaired in egress. Whether wild type
p23 protease, which cleaves parts of the cytoplasmic
intermediate filament network, directly or indirectly
facilitates virus exit rather than cell lysis is not known.75,76

ENTRY, DISASSEMBLY AND NUCLEAR

IMPORT
Each virion leaving an infected cell carries at least 10
copies of inactivated cysteine protease.36,55,57 To initiate
a new round of infection, extracellular virus attaches with
high affinity to a target cell (see Figure 3). Recently, a
primary receptor of the immunoglobulin gene family,
CAR (coxsackie virus, adenovirus receptor) has been
identified as a specific determinant recognising the distal
fibre head domain of ad2 and ad5.77,78 An alternative
receptor has also been identified as MHC class I.79 Entry
is facilitated by binding of penton base to a secondary
receptor, alpha v-beta 3/5 integrins, as demonstrated by
cDNA transfection experiments in integrin-deficient cell
lines.80 Virus endocytosis then occurs through clathrincoated vesicles8183 requiring the arginineglycine
aspartate (RGD)-domain of penton base.8084 Contact of
penton base with fibronectin/vitronectin binding
integrins somehow facilitates capsid penetration of the
endosomal membrane.85,86 Penetration is enhanced by
acidic endosomal pH,87,88 but the precise mechanism is
unknown. Penetration at low multiplicity of infection
occurs approximately 15 min after virus internalisation, as
suggested by neutralisation of acidic endosomes with
lysosomotropic amines.86 At high doses of virus, penetration can occur in the absence of acidic endosomal pH as
suggested by (89) and our own unpublished results. In
fact, the first step in the virus disassembly programme,
fibre shedding, occurs in the absence of acidic endosomes,
e.g. in cells treated with the ionophore monensin.86
Capsid dismantling is a stepwise process starting with
fibre release at the time of virus endocytosis. Half of the
incoming viruses lose their fibres within 10 min of
internalisation initiation.86 After 30 min, more than 90%
of the virions are fibre-less. When virus interactions with
integrins are disturbed with RGD peptides or antiintegrin antibodies, fibres are not released and the virus
remains at the cell surface (Greber, unpublished data).
Whether quantitative fibre detachment is necessary for
virus endocytosis is not known at the moment. Interestingly, the protease mutant virus ts1 does not lose all of
its fibres, but is internalised with the same efficiency and
kinetics as wild type virus.55 It is possible that fibre
shedding occurs also in ts1, but only at those vertices
which engage with surface receptors. The next proteins
Rev. Med. Virol. 8: 213222 (1998)

218

to be released after the fibres are the capsid-stabilising

proteins IIIa and VIII.86 Protein IX, a cementing factor
which keeps the hexons together is removed between 30
and 60 min after entry, long after the virus has reached
the cytosol. Removal of protein IX, a capsid stabilising
component, coincides with, but is not sufficient for capsid
disassembly.
To liberate the DNA and dissociate the capsid reactivation of the capsid-resident cysteine protease p23 must
also occur. As discussed above, p23 activity can be
abolished in vitro by alkylating agents provided that the
active centre cysteine or the cysteine accepting the
activating peptide is present in the reduced thiol state. If
freshly isolated viruses are treated with alkylating agents,
virus-mediated gene expression is reduced several thousand fold.90 Alkylated viruses can bind, penetrate and
localise to the nuclear membrane with similar efficiencies
as wild type virus, but are not able to release the
genome.55 Wild type viruses extensively purified in the
absence of reducing agents or treated with oxidising
agents, such as diamide, retain full infectivity and are not
affected by a large variety of alkylating agents. Viruses
become sensitive to alkylation only if they are treated for
a short time with low concentrations of a reducing agent,
such as dithiothreitol (DTT) or glutathione. p23 of
reduced virions but not oxidised virions is heavily
modified with radiolabelled NEM, a strong sulphhydryl
alkylating agent. p23 extracted from reduced virions has
a faster mobility in non-reducing SDS gels than p23 from
extensively purified or oxidised particles suggesting a
redox-dependent conformational change in the protease.
Such a change would be consistent with either the
dissociation of the activating peptide or the reduction of
intramolecular disulphide bonds. Oxidation, on the other
hand, is not required for virion infectivity, since fully
infectious virus particles can be isolated from cells in the
continued presence of reducing agents. Extracellular oxidation can thus be a safeguard for the virion against
uncontrolled proteolysis.
Encountering reducing milieu in the non-infected cell is
necessary, but not sufficient for p23 reactivation. The
incoming virus must also contact cell surface integrins.55
In a normal infection p23 stimulates the degradation of
the internal protein VI. Protein VI degradation cannot be
triggered on the cell surface in the presence of DTT at
physiological temperature, if proper contact of virus
penton base with integrins is inhibited, e.g. with RGD
peptides. That protein VI degradation is mediated by the
viral p23 rather than cellular proteases is supported by
several lines of evidence. First, protein VI contains a
degenerate consensus site for p23 cleavage as deduced
from the DNA sequence.91 Second, recombinant protein
VI precursor can be readily degraded by activated
recombinant protease.71 Third, protein VI degradation
can occur in two compartments, the cytosol or endosomes. Predominantly cytosolic degradation of protein
VI occurs during normal infection with a half time of
about 20 min, when most of the virions have escaped
from endosomes.86 If endosomal escape, e.g. at low
multiplicity of infection, is inhibited by lysosomotropic
 1998 John Wiley & Sons, Ltd.

U. F. GREBER

agents, such as ammonium chloride or monensin, which

neutralise vesicular pH, protein VI degradation occurs at
the same efficiencies as without inhibitors. This result is
consistent with the neutral pH optimum of p23.61 Fourth,
endosomal or lysosomal proteases are unlikely mediators
of protein VI degradation, since loading cells with
lysosomal protease inhibitors, such as leupeptin, has no
effect on protein VI degradation, but completely stops
the low level of hexon degradation. Lysosomal degradation of hexon in the protease deficient ts1 virus readily
occurs in the absence of any protein VI precursor
breakdown strongly supporting the notion that protein
VI degradation is catalysed by p23.55
From the averaged adenovirus capsid structure,
Burnett and coworkers predicted that protein VI links the
peripentonal hexons to the DNA core.29 In agreement
with this view are results from blot binding experiments
suggesting that protein VI binds to double-stranded
DNA and also to hexon.70,92 It is thus possible that
integrin binding to penton base induces a conformational
change in the peripentonal capsid region which renders
protein VI susceptible to proteolysis.
It appears that p23 mediated proteolysis of protein VI
weakens, but does not dissolve the cytosolic capsid. In
fact, quantitative electron microscopy of thin sectioned
epitheloid HeLa cells indicated that both, proteaseinactivated and wild type virus capsids specifically attach
to the nuclear pore complexes of infected HeLa cells. Of
the particles located within two capsid diameters at the
nuclear envelope, 23% of protease-inactivated and 29%
of native capsids, respectively, are associated with nuclear
pore complexes.55 Interestingly, only partially disassembled capsids, but not so called empty capsids are
readily detected in association with nuclear pore
complexes (NPCs) suggesting that capsids devoid of
contents are either unstable or difficult to detect.
Besides protein VI degradation, import of viral DNA
and associated protein VII into the nucleus also requires
contact between the weakened capsid and cytosolic
elements of the NPC.93 Specific inhibitors of O-linked
glycoproteins of the NPC, such as the RL1 antibody or
wheat germ agglutinin, microinjected into living cells
block virus attachment to the nuclear membrane and
inhibit capsid disassembly. It is possible that surfaceexposed determinants of either hexon or penton base are
involved in capsid attachment to the NPC. Protein IX is
less likely to be involved in such interactions, since it
does not appear to be located at the capsid surface.94
Whether capsid disassembly is directly triggered by an
integral NPC component or indirectly, together with
peripherally attached cytosolic components is not known.
It is also unclear if protease of incoming virus particles
mediates proteolysis of cellular proteins, such as actin or
fibrin, as suggested by in vitro cleavage assays.60
While docking and capsid dissociation occur independently of nuclear envelope calcium levels, nuclear DNA
import is inhibited if luminal calcium is depleted by
ionophores or a calcium ATPase inhibitor, thapsigargin.93
These results are in agreement with earlier studies in
cultured somatic cells and oocytes demonstrating a
Rev. Med. Virol. 8: 213222 (1998)

ASSEMBLY AND DISASSEMBLY

calcium-dependent function of the NPC.9597 The data

suggest that the nuclear envelope can function as a
regulatory barrier in nucleo-cytoplasmic trafficking of
viral and cellular macromolecules. Whether the terminal
protein, p55, which is covalently attached to the DNA
and contains a transferable classical nuclear localisation
signal (NLS),98 is somehow involved in threading the
linear DNA through the NPC is, however, not yet
known. Inside the nucleus, the terminal protein somehow
tethers the DNA to the nuclear matrix,99 perhaps in close
proximity to a nuclear subregion characterised by the
presence of nuclear domain 10 (ND10) antigens.100 It is
possible that proteins V, VII and , which are noncovalently associated with virion DNA, are stripped off
the nuclear DNA to allow the formation of histonecontaining nucleosomes for efficient transcription and
replication.

CONCLUSIONS
It is clear that assembly and disassembly of a complex
virus capsid, such as the adenovirus, depends on many
viral and cellular factors (see Figure 3). One common
regulator of both assembly and disassembly has recently
been identified as the virus-encoded cysteine protease
p23. p23 carries out most of its action within the viral
particle. It receives two temporal activations. The first
activation occurs late in assembly by one of its own
substrates. The second activation takes place early in
disassembly by a two-fold trigger, binding of capsid to
integrin receptors (or a consequence thereof), and the
reducing milieu of the cytoplasm. Understanding in even
greater detail the differences between assembly and
disassembly pathways of this complex virus will be
essential for a successful therapeutic use of either
complete virus particles or any subviral particles.

219

6.
7.
8.
9.
10.

11.
12.
13.

14.
15.
16.

ACKNOWLEDGEMENTS
I thank Dr R. Stidwill and Dr M. Suomalainen for helpful
comments to the manuscript. Work in the authors
laboratory has been supported by the Kanton Zrich and
a grant from the Swiss National Science Foundation.

17.
18.

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