endocytosis of adenovirus and adenovirus capsid proteins

www.elsevier.com/locate/addr

Advanced Drug Delivery Reviews 55 (2003) 1485–1496

Endocytosis of adenovirus and adenovirus capsid proteins

Lali K. Medina-Kauwe*

Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine,

Institute for Genetic Medicine, 2250 Alcazar Street, CSC240, Los Angeles, CA 90033, USA

Received 5 July 2003; accepted 30 July 2003

Abstract

Key proteins of the icosahedral-shaped adenovirus (Ad) capsid mediate infection, and interact with cellular proteins to

coordinate stepwise events of cell entry that produce successful gene transfer. Infection is mediated predominantly by the

penton and fiber capsid proteins. The fiber initiates cell binding while the penton binds integrin coreceptors, triggering integrin-

mediated endocytosis. Penton integrin signaling precedes viral escape from the endosomal vesicle. After cell binding, the virus

undergoes stepwise disassembly of the capsid, shedding proteins during cell entry. Intracellular trafficking of the remaining

capsid shell is mediated by the interaction of naked particles with the cytoskeleton. The capsid translocates toward the nucleus,

with the majority of capsid proteins accumulating at the nuclear periphery, while viral DNA and associated protein VII are

extruded through the nuclear pore. This discussion will encompass the current knowledge on Ad cell entry and trafficking, with

an emphasis on the contribution of Ad capsid proteins to these processes. A greater understanding of the highly effective Ad cell

entry pathway may lend itself to the development of safer drug and gene delivery alternatives utilizing similar pathways.

D 2003 Elsevier B.V. All rights reserved.

Keywords: Adenovirus; Capsid; Penton; Fiber; Endocytosis

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1486

2. Cell binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1487

2.1. Fiber. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1487

2.2. Penton . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1488

3. Cell entry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490

3.1. Endocytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490

3.2. Endosomal lysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1490

4. Intracellular trafficking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1491

5. Nuclear delivery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1492

6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1492

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1493

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1493

0169-409X/$ - see front matter D 2003 Elsevier B.V. All rights reserved.

doi:10.1016/j.addr.2003.07.010

* Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Davis Building, Room 5092, 8700 Beverly Boulevard, Los Angeles,

CA 90048, USA. Tel.: +1-310-423-7339; fax: +1-310-423-7308.

E-mail address: [email protected] (L.K. Medina-Kauwe).

L.K. Medina-Kauwe / Advanced Drug Delivery Reviews 55 (2003) 1485–14961486

1. Introduction

Adenoviruses (Ads) comprise a family of non-

enveloped double-stranded DNA viruses containing

a genome of about 36 kb. Human Ad includes at least

50 known serotypes grouped into six distinct sub-

groups (A through F) with different pathophysiolo-

gies, ranging from gastrointestinal to ocular to

respiratory infection. Ad serotypes 2 and 5 (Ad2

and Ad5), belonging to subgroup C, are very closely

related and widely studied as gene therapy vectors.

Despite the potent immune response elicited by Ad

vectors [1,2], the highly efficient cell entry mecha-

nism of the virus remains a desirable feature for gene

delivery to both dividing and nondividing cells [3,4].

Key to infection efficiency is the outer protein coat, or

capsid, of the virus itself, as realized during the

development of targeted gene delivery systems [5].

One of the earliest targeted systems, utilizing trans-

ferrin chemically linked to polylysine for DNA trans-

port [6], produced high levels of receptor binding and

internalization but relatively low transgene expres-

sion. The co-uptake of transcriptionally inactive Ad

greatly augmented transferrin-mediated gene transfer,

indicating that the viral capsid itself promotes cyto-

solic entry processes in the absence of viral gene

expression [7–9].

What features of the Ad capsid enable effective

gene transfer? Three proteins make up the icosahe-

Fig. 1. Schematic of the Ad capsid. (A) Whole capsid identifying fiber,

showing homotrimeric fiber bound to homopentameric penton base. (C) Fi

not drawn to scale.

dral-shaped outer capsid shell (Fig. 1A). A structural

protein called the hexon comprises the majority of

the capsid [10]. Capping each vertex of the Ad

icosahedron is a penton base, which is comprised

of a homopentameric ring of proteins. An antenna-

like, homotrimeric fiber protein protrudes from each

penton base, forming penton fiber capsomers (Fig.

1B) [11]. Elsewhere in the literature, the terms

‘penton base,’ ‘penton capsomers,’ and ‘penton’ are

used interchangeably and may include the presence

of the fiber. Here, for the sake of clarity, ‘penton

base’ refers to the penton protein only, whereas

‘penton fiber capsomers’ designate penton base

bound by the fiber.

Ad infection is mediated predominantly by the

penton and fiber capsid proteins. As summarized

here, these proteins interact with cellular factors to

coordinate key events that permit the passage of viral

particles into the cell and, ultimately, to the nuclear

periphery for gene transfer. While the penton and

fiber proteins alone do not act autonomously to

mediate the transport of Ad DNA to the nucleus

during infection, this review will highlight the im-

portant roles that these proteins play in enabling

efficient Ad gene transfer. More importantly, these

activities can be taken into consideration in the

development of gene and drug transport systems that

may incorporate viral proteins for improved delivery

of therapeutics.

penton base, and hexon. (B) Enlargement of circled region in (A),

ber monomer, identifying tail, shaft, and knob domains. Pictures are

L.K. Medina-Kauwe / Advanced Drug Delivery Reviews 55 (2003) 1485–1496 1487

2. Cell binding

2.1. Fiber

The fiber is responsible for initial cell attachment,

and consists of three domains: tail, shaft, and knob

(Fig. 1C) [12,13]. The tail comprises the first 17

residues from the amino (N)-terminus and is respon-

sible for noncovalent attachment of the fiber to the

capsid penton base. The shaft consists of 22 structural

repeats downstream from the tail, which form a

protease-resistant h-spiral structure [14]. The remain-

ing carboxy (C)-terminal segment folds into a globu-

lar knob that contains a receptor-binding domain and a

trimerization domain [15,16].

The fiber of subgroup C Ad, which includes Ad

serotypes 2, 5, and 12, binds with high affinity

(Kd = 1.7 nM) [17] to the coxsackievirus–adenovirus

receptor (CAR) cell surface protein [18]. This 46-kDa

single transmembrane protein bears structural similar-

ity to immunoglobulin (Ig) gene superfamily proteins,

especially cadherins. Its 222-amino acid (aa) extracel-

lular domain contains two Ig-like regions, designated

D1 and D2, that both interact with the Ad2 and Ad12

fiber knob, although D1 alone is sufficient for binding

[19]. The cytoplasmic and transmembrane domains,

on the other hand, are dispensable for viral infection,

as truncated receptors lacking these domains still

support Ad and coxsackievirus transduction [20].

The cellular function of CAR likely requires these

domains, as the cytoplasmic regions of human and

mouse CAR share 95% identity, suggesting that these

receptors share common intracellular interactions and

signaling [20]. Structural and mutational studies on

the fiber knob show that each knob trimer binds three

CAR molecules, each at a deep facet formed between

adjacent knob monomers, thus evading antigenic

neutralization of the receptor-binding site [21,22].

While receptor clustering is a common strategy for

inducing intracellular signaling, it is unclear why

clustering of CAR would be a necessary Ad strategy

given that the cytoplasmic domain is not required for

infection.

CAR is expressed in the brain, liver, heart,

lungs, and kidneys [23], and specifically localizes

with avh3 and avh5 in cardiomyocyte intercalated

discs and sarcolemma [24]. The relatively high

cardiac expression of CAR during embryonic de-

velopment, decreased expression in the adult, and

upregulation in diseased tissue suggests that CAR

may act as a pathfinder protein, directing organ

formation during embryogenesis and re-expressing

this embryonic pattern after cytopathic damage

[24,25].

In cultured cells, CAR induces the formation of

cell aggregates and accumulates at cell–cell contacts,

forming homotypic interactions between extracellular

CAR domains of apposing cells [26,27]. Its targeting

to basolateral surfaces of polarized epithelia and

colocalization with ZO-1 strongly indicates that it is

a component of the tight junction, as illustrated in

Fig. 2A [26,28]. If so, how, then, does Ad access this

protein for infection? It has been shown previously

that the affinity of the fiber knob for CAR is higher

than that of CAR for itself, thus it is possible for Ad to

disrupt CAR dimers [14,19]. It is also possible that Ad

exploits breaches in cell–cell contacts to gain access

to the basolateral surface where it can further interact

with integrins for cell entry, as illustrated in Fig. 2B.

The need for the fiber protein to open up these barriers

is demonstrated during Ad infection of airway epithe-

lia, in which overproduced fiber released from lysing

cells can bind CAR, thus disrupting junctional integ-

rity and allowing the release of viral progeny onto the

apical surface [28].

No study thus far indicates that CAR undergoes

receptor-mediated endocytosis after Ad binding

[29,30]. This does not necessarily mean, however,

that the fiber serves merely in the capacity of docking

the virus to the cell surface. Indeed, events taking

place immediately after cell binding suggest that the

fiber may regulate the cell entry, endosomal lytic, and

intracellular trafficking steps of Ad infection. For

example, after cell binding, the fiber is released from

the Ad2 capsid while still on the cell surface, a step

that requires coactivation of integrins by the penton

base and actin polymerization [31,32]. The tempera-

ture-sensitive mutant virus, ts1, which fails to release

fibers, binds and internalizes into cells but fails

vesicular escape, thus indicating that key steps of

capsid dismantling, beginning at the cell surface, are

required for proper infection [33,34]. This shedding

may provide greater access of the penton to integrins,

keep tight junctions open to allow virus escape, or

alternatively allow the virus to infect non-CAR-

expressing cells.

Fig. 2. Schematic of the Ad infection pathway. (A) Homotypic binding of CAR at tight junctions. (B) Interaction of fiber knob with CAR trimer.

(C) Interaction of penton base with integrins. Fibers have already begun to be released from the capsid at this step. (D) Clathrin-coated pit

formation at integrin Ad binding sites. (E) Clathrin-coated vesicle formation containing Ad. (F) Further dismantling of capsid and acidification

of endosome. (G) Interaction of naked capsid with microtubules and dynein motors. (H) Docking at nuclear pore complexes and passage of viral

DNA through nuclear pores. Objects in drawing are not drawn to scale.


Finally, although CAR has become widely studied

as a viral receptor, the subgroup C Ad fiber may

recognize alternative receptors to initiate infection.

The Ad5 knob interacts with MHC I through a

region that does not overlap with the CAR-binding

site [35], while a KKTK sequence in the Ad2 and

Ad5 fiber shafts recognizes heparan sulfate proteo-

glycans [36].

2.2. Penton

Fiber–CAR interaction is followed by the binding

of the penton proteins to avh3 or avh5 integrins (Fig.

2C) [17] through a conserved Arg–Gly–Asp (RGD)

consensus motif [37–40]. Cryo-EM shows the RGD

motifs on protrusions that project away from the

central axis of the penton base homopentamer, from

which the fiber shaft protrudes [41]. Weak densities at

these protrusions on the Ad2 penton indicate high

mobility and suggest that the RGD sites lie at the ends

of flexible loops, thus aiding access to integrins. With

the fiber bound, IgG recognition of the RGD motifs is

sterically hindered, thus preventing antibody neutral-

ization of RGD [42]. In fiberless Ad3 and Ad5

capsids, however, these protrusions point slightly

inward toward the central axis of the pentamer, thus

indicating that the penton undergoes structural

changes when the fiber is released [43,44]. These

findings suggest that either fiber release triggers

structural changes in the penton that are required for

proceeding to subsequent steps of cell entry or,

conversely, that penton ligation of integrins induces


structural changes that permit fiber release. Given the

integrin-binding requirements for fiber release dis-

cussed earlier, the latter explanation may be the most

likely scenario.

The lower-affinity interaction (Kd = 55 nM) of

pentons with integrins is compensated by increased

avidity due to pentamer formation. This promotes

integrin clustering and enhances downstream cellular

signals through enhanced integrin ligation [45].

Cryo-EM imaging of soluble integrin molecules

Fig. 3. Immunofluorescence and confocal microscopy of HeLa cells treat

(LLAA), or fiber (WTF) protein. (A–C) Cells were exposed to 20 Ag of PB

then warmed to 37 jC for (A) 0 min, (B) 5 min, or (C) 45 min before fix

(green) and actin (red), following standard procedures [90]. In (A), the fluo

at the cell surfaces at 4 jC. In (B), PB appears in aggregated foci at the ce

surrounds the nuclear periphery. (D) Immunostain of HeLa cells after intern

in scattered foci throughout the cytoplasm, suggesting that the intracellular

after exposure to (E) 40 Ag of WTF, or (F) 20 Ag of PB for 1 h at 37 jmagnification bar, f 10 Am.

bound to the Ad12 penton base shows a cleft formed

by av and h5 subunits into which the RGD loop fits.

The soluble integrin molecules form a ring of five

receptor proteins bound to each homopentamer.

Clustering of integrins activates signaling that trig-

gers cytoskeletal changes [46]. The precise spatial

arrangement of the ligand may distinguish the type

of signal, as RGD-containing oligopeptides do not

elicit signaling [32]. This strategy is exploited by the

unrelated foot-and-mouth disease virus (FMDV),

ed with soluble recombinant wild-type penton (PB), mutant penton

for 2 h at 4 jC in serum-free medium supplemented with 3% BSA,

ation in 4% paraformaldehyde. Cells were coimmunostained for PB

rophores overlap at the cell peripheries, indicating that PB is retained

ll peripheries, suggesting that the proteins are clustered. In (C), PB

alization of 20 Ag of LLAA mutant. PB (indicated in yellow) appears

translocation is disrupted. (E–F) Actin staining (red) of HeLa cells

C. Arrowheads point to cytoskeletal extensions in (E). n = nucleus;


which bears an identical spatial arrangement of viral

capsid proteins that also bind integrin and induce

clustering [47,48].

Recombinant pentons also appear to accumulate

in clustered foci at the cell periphery, thus the intact

capsid may not be a requirement for receptor aggre-

gation. At 4 jC, a temperature that promotes receptor

binding but not endocytosis, soluble recombinant

penton (PB) is bound at the periphery of HeLa cells

(Fig. 3A). After warming to 37 jC for 5 min, cell-

bound PB proteins begin to aggregate at cell surfaces

(Fig. 3B), suggesting that receptors cluster after

ligation. As homopentamer formation of recombinant

soluble pentons has been demonstrated previously

[40,49], it appears to be sufficient here to induce

focal clustering in the absence of the rest of the

virus.

3. Cell entry

3.1. Endocytosis

Cytoskeletal changes immediately take place after

Ad binding, and are manifested as filipodial exten-

sions, followed by lamellipodia formation and mem-

brane ruffling [50]. Whereas such Ad-induced

cytoskeletal changes are attributed to integrin ligation

by pentons, a soluble recombinant fiber protein

(WTF) elicits the extension of many more filipodia

in HeLa cells (Fig. 3E) compared to an equimolar

concentration of PB (Fig. 3F). The integrin receptor

clustering resulting from penton binding enhances

intracellular signaling and local formation of cla-

thrin-coated pits. An NPXY motif, matching a tyro-

sine-based consensus sequence for endocytic sorting

[51,52], is found in the cytoplasmic tails of h3 and h5

integrin subunits [53], and mediates the localization of

receptors to coated pits (Fig. 2D) [54]. Dynamin, a

100-kDa cytosolic GTPase associated with clathrin-

coated pits, regulates clathrin-mediated endocytosis

by enabling the constriction and budding off of coated

pits into coated vesicles (Fig. 2E) [55,56]. Dominant

negative dynamin reduces Ad2 internalization by up

to 70% [57], thus supporting a model of viral cell

entry into clathrin-coated vesicles.

Both Ad2 and recombinant penton binding to cells

activate phosphatidylinositol-3-OH kinase (PI3K),

which, in turn, activates Rac and CDC42 GTPases,

thus inducing actin polymerization and viral endocy-

tosis into clathrin-coated vesicles [50,58]. Viral endo-

cytosis is selectively mediated by intact actin mic-

rofilaments, as cytochalasin D, which disrupts actin,

prevents viral uptake, whereas nocodazole, which dis-

rupts microtubules, does not [59]. Endocytosis appears

to be rapid, as up to 30% of bound Ad is internalized

within 15 min [17].

3.2. Endosomal lysis

The Ad2 capsid continues to dismantle during viral

entry, shedding more fiber proteins and exposing

more of the penton protein [33]. As the virus-contain-

ing endosome matures, the vesicular pH drops due to

the acidification by proton pumps (Fig. 2F). At pH

6.0, the virus penetrates the endosomal membrane and

escapes to the cytosol, thus evading degradation by

lysosomal enzymes [3,60]. The endosomal lytic ac-

tivity of the virus is attributed to the penton, as studies

show that the penton undergoes a conformational

change at low pH, exposing hydrophobic regions that

bind nonionic detergents [61]. Moreover, a blocking

antibody against the penton inhibits endosome escape

[62]. This activity coupled to integrin-mediated cell

entry has been exploited for the creation of penton-

based nonviral gene delivery vectors [49,63–65],

suggesting that recombinant soluble pentons may

follow the same translocation pathway as the whole

virus.

Additional factors contribute to successful endo-

some escape. At low pH, the virus binds selectively to

avh5 integrins, which are key to enabling penetration

to the cytosol and which support twofold to fourfold

higher levels of Ad gene transfer than avh3 [66,67].

The cytoplasmic domain of the h5 subunit contains a

TVD motif that selectively mediates membrane pen-

etration [66]. Cellular proteins that may bind this

motif have not yet been identified. While these find-

ings, taken together, imply that an acidic environment

triggers endosome lysis, this criterion remains ambig-

uous in the light of findings that agents that raise

vesicular pH do not prevent Ad2 infection [68].

The induction of macropinocytosis also correlates

with Ad escape from endosomes, as the inhibition of

macropinosome formation with amiloride reduces

endosomal escape by nearly 50% [69]. Macropino-


somes form by the pinching off of large invaginations

formed from membrane ruffles and comprise a non-

clathrin-mediated pathway usually triggered by

growth factor stimulation [54]. Ad-induced macro-

pinocytosis is av integrin-dependent. It also requires

F-actin and protein kinase C (PKC) activation, but

does not require viral uptake, implying that signaling

from the cell surface triggers both macropinosome

formation and endosome leakiness.

Additional signaling required for endosomal lysis

is the activation of PKC, as PKC inhibition does not

prevent endocytosis but does inhibit endosome escape

[31,69]. The role of PKC in endosome lysis is unclear,

and could either combine with integrin signaling to

activate lysis, or mediate the trafficking of virus-

containing endosomes to enable proper maturation

and acidification for vesicular release.

The fiber also influences cytosolic entry by regu-

lating the timing of endosome escape, as shown by

molecular swapping of fibers from different sub-

groups [70]. Ad5 (subgroup C) is released from

endosomes early after endocytosis at pH 6.0, when

the vesicle is still near the cell periphery, while Ad7

(subgroup B) is retained longer in the endosome and

released when the vesicle is near the nucleus, at pH

5.5 [71]. Exchange of fibers switches these patterns of

endosome escape, so that an Ad5 capsid pseudotyped

with an Ad7 fiber adopts the trafficking pattern of

Ad7 and vice versa [70]. These findings suggest that

the fiber may act as a pH sensor during endocytic

progression, thus dictating the temporal and spatial

localization of the capsid upon vesicle exit. The

intriguing possibility that the fiber itself lyses the

endosomal membrane is supported by Zhang et al.

[21], who described the membrane translocation of a

peptide (Peptide I) comprised of the Ad3 fiber tail

sequence appended to a polylysine sequence.

Finally, the Ad protease greatly influences viral cell

entry and endosome escape. The L3/p23 protease

cleaves six viral proteins, an activity required to

produce mature, infectious virus [72]. The protease

is packaged into the capsid and inactivated after

capsid release from the cell and into an oxidizing

environment, but reactivated after entry into acidified

endosomes [33,73]. A temperature-sensitive mutant

Ad, ts1, encodes misfolded protease that is not pack-

aged into capsids [74,75]. The ts1 mutant particles

contain unprocessed proteins, fail to release fibers

during viral uptake, and do not lyse endosomes

[33,34]. Dismantling of the viral capsid, especially

fiber release, appears to play a significant role in cell

entry and endosomal lysis.

4. Intracellular trafficking

After cell entry and endosome escape, Ad capsids

translocate toward the nucleus and accumulate at the

nuclear periphery. Signaling is required for this traf-

ficking, as microinjected Ad is immobile, while infec-

tion triggers trafficking to the nuclear periphery [59].

Motilities are mediated by at least two signaling path-

ways [76]. The first is an integrin-dependent pathway

mediated by cAMP-dependent PKA. Intracellular

cAMP levels regulate microtubule-dependent vesicle

transport [77,78], and are likewise critical for regulat-

ing viral traffic. Integrin binding is required to activate

PKA in HeLa cells, and PKA inhibition both prevents

nuclear targeting of Ad 2 and enables net movement

toward the cell periphery. The second pathway is

integrin-independent and requires the activation of

p38/MAPK to suppress motility directed toward the

cell periphery. Thus, both pathways appear to work in

concert to promote movement toward the nucleus.

Trafficking studies on Ad2 and Ad5 show that

nocodazole, which disrupts intact microtubules, has

no effect on cell entry or endosome lysis but prevents

viral translocation to the nucleus [59]. After endosome

escape, the naked virus particle rapidly translocates

along microtubule tracks at rates of up to 2 Am/s in

saltatory movements with a net motility toward the

nucleus (Fig. 2G) [59,79]. Microtubule polarity may

permit proper particle navigation in the cytoplasm, as

the fast-growing ‘‘plus’’ ends, to which tubulin dimers

are added, are typically found at the cell periphery

whereas the slow-growing ‘‘minus’’ ends are found at

the microtubule organizing center (MTOC), at the

center of the cell [80–82]. The microtubule-dependent

motor, dynein, which typically supports minus-end

directed movement, is a likely candidate for Ad trans-

location. Indeed, the disruption of dynein motors by

either expression of dynamitin [59,83] or a function-

blocking antibody against dynein [84,85] inhibits Ad

nuclear targeting. Particles navigating along intact

microtubules eventually accumulate at the MTOC,

which is perinuclear in cultured cells [86]. The move-


ment of subgroup B Ad may also require dynein

motors, as Ad7 exhibits rapid motility and perinuclear

accumulation after endosome escape, similar to sub-

group C Ad, although this movement occurs later in

infection due to its prolonged endosomal retention

[71]. The ts1 mutant, which does not escape the endo-

some, remains randomly distributed in the cytoplasm

rather than accumulate at the perinucleus, and is likely

targeted to lysosomes [59]. These findings support the

idea that cytoplasmic, rather than endosomal, particles

interact with microtubule-dependent motors. As dy-

nein motors typically transport cargo enclosed in

vesicles [87–89], the direct interaction of the naked

particle or capsid components with dynein is an

intriguing possibility that has yet to be demonstrated.

After endosome escape, f 80% of the capsid is

still intact [3] and still contains hexon and penton

proteins. Thus, the hexon, penton, or both may

mediate translocation toward the nucleus. Recombi-

nant PB accumulates around the nucleus by 45 min

after cell entry in a pattern similar to whole Ad (Fig.

3C). While direct interaction of the penton with

microtubule-associated motors remains to be seen,

indirect findings suggest that the penton may seques-

ter host trafficking machinery after internalization.

Rabbit lacrimal gland acini exhibit a reduction in

stimulated protein secretion and a loss of Rab3D-

enriched mature secretory vesicles after binding and

uptake of PB, but not recombinant knob protein [90].

Apical actin depletion, microtubule bundling, and

disorganization accompany these changes—effects

not seen after exposure to the knob. Candidate motifs

that may mediate this effect include a region on the

penton that becomes exposed after fiber release, and

contains the sequence 253SRLSNLLG, identified as

the fiber-binding site [91]. This sequence matches the

well-characterized dileucine (or LL) motif, a sorting

signal found in numerous trafficking proteins [51,52].

Dileucine motifs directly bind clathrin-associated

adaptor protein (AP) complexes that mediate endo-

cytic and secretory pathways. Ad-mediated sorting in

endosomes and/or trafficking to the nucleus may

therefore involve competitive recruitment of host AP

complexes. Indeed, a mutation to this sequence in PB

alters its trafficking in HeLa cells (Fig. 3D). After cell

binding and uptake, control pentons surround the

nucleus whereas dileucine mutants exhibit disorga-

nized localization.

5. Nuclear delivery

Upon convergence at the perinuclear envelope,

capsids dock at the nuclear pore complex (NPC) and

undergo further dismantling [92]. Final dissociation

of the capsid, and subsequent nuclear import, rely on

the L3/p23 protease to degrade internal protein VI

and, as expected, the ts1 mutant does not undergo

disassembly or nuclear import [33]. The majority of

capsid proteins, including the hexon and penton,

remain at the perinuclear envelope [33]. Inhibitors

of O-linked NPC glycoproteins inhibit docking and

dismantling, suggesting that the NPC has disman-

tling activity [92]. This activity is possibly mediated

by flexible cytoplasmic fibrils of O-linked NPC

glycoproteins [93]. These same inhibitors prevent

the import of nuclear localization sequence (NLS)-

containing proteins and ribonucleoproteins [94–96]

and likewise prevent the import of DNA-associated

protein VII. After complete dismantling, viral DNA

and protein VII are extruded through the nuclear

pores (Fig. 2H) [3]. Intracellular calcium depletion

inhibits nuclear targeting of NLS-containing proteins

[97,98]. Likewise, nuclear import of viral DNA and

protein VII is prevented when internal calcium stores

are depleted after endosome escape [92]. It is possi-

ble that calcium induces conformational changes in

the NPC that permit passage of macromolecules

[99].

6. Conclusions

From the current scope of literature on Ad infec-

tion emerges a picture of the steps involved for Ad

cell entry and intracellular translocation. Many of

these steps require interaction of viral proteins with

host proteins to elicit specific signaling. Such signal-

ing produces cellular responses that enable viral

passage. As the redundancy of certain pathways in

other viruses is not uncommon, an understanding of

Ad infection mechanisms may contribute to improve-

ments in developing novel antiviral therapies. Impor-

tantly, serious concerns of toxicity and immunogenic-

ity have prompted widespread efforts to improve the

safety of Ad as a therapeutic gene delivery vector.

Nonviral alternative vectors that utilize Ad transloca-

tion pathways to obtain effective gene or drug transfer


could be developed. As such vectors utilizing Ad

capsid proteins are currently in development, an

understanding of the contribution these proteins make

toward infection as well as molecular and cellular

requirements for translocation will aid in making

improvements to these systems.

Acknowledgements

Many thanks to Sarah Hamm-Alvarez for many

helpful discussions on this topic, and to Xinhua Chen

and Meghan Maguire for contributions to the author’s

work. Many thanks also to Larry Kedes and Nori

Kasahara for supporting the author’s work, and the

present and former members of the Hamm-Alvarez,

Kedes, and Kasahara laboratories for their useful

discussions. Work in the author’s laboratory was

supported by grants from the USC Liver Center (5

P30 DK048522-09) and the USC Cancer Center

(American Cancer Society Institutional Research

Grant Individual Allocation).

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