Biology of Adenovirus Cell Entry

Biology of Adenovirus Cell Entry: Receptors, Pathways, Mechanisms

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Stefania Luisoni, Urs F. Greber Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland

1.  Introduction The family of Adenoviridae is composed of a large number of pathogens that cause respiratory, ocular, blood-borne, and intestinal infections and can heavily affect immunecompromised individuals.1,2 Adenoviruses (AdVs) are widely used vectors for virotherapies (http://www.wiley.com/legacy/wileychi/genmed/clinical/). These nonenveloped nanoparticles can be grown to high titers and purified to near homogeneity under good manufacturing practice.3,4 Adenoviruses are popular gene delivery agents because they transduce both dividing and quiescent cells efficiently, have high physical and genetic stability, and occur in a large number of variants yielding high application flexibility. The infection biology of certain AdV types has been elucidated in sufficient detail to grant clinical applications. In particular, deep mechanistic insight has been gained with human AdV (HAdV) types of the species B and C, and members of the HAdV-C are widely used in oncolytic therapy. HAdVs E4 and B7 have been used successfully as a vaccine in enteric applications for military personnel.5,6 The therapeutic potential of AdV-based vectors for clinical applications is broad and includes novel vaccination strategies and oncolysis.7,8 A major challenge for the field has been to better understand features that make AdVs so highly immunogenic and distinct features that render AdV vectors the most popular cancer cell killers. Such insights will provide a basis to improve vectors: for example, to increase their circulation time and better target them to cells of interest in clinical applications.9

2.  Entry Pathways: Impact of Capsid Proteins Virus entry determines the efficacy of virotherapies, disease onset, and progression. Adenovirus entry is best characterized in epithelial cells, although entry into immune cells is of emerging importance (for reviews, see Refs 10, 11). Adenovirus entry occurs in sequential steps, including virus attachment to target cells, signaling, endocytosis, endosomal escape, transport through the cytosol, separation of the viral genome from the capsid, and delivery of the genome into the nucleus (reviewed in Refs 12–16). A schematic summary of generic HAdV entry is depicted in Figure 1. Even minor defects or enhancements in the entry process, for example by soluble factors such as serum factors, have an impact on viral gene expression or innate immunity responses against the incoming virus. Adenoviruses are icosahedral particles of pseudo T = 25 triangulation, in which each facet has 12 trimers of the major coat protein hexon. The hexons contain exposed Adenoviral Vectors for Gene Therapy. http://dx.doi.org/10.1016/B978-0-12-800276-6.00002-4 Copyright © 2016 Elsevier Inc. All rights reserved.

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Figure 1  Schematic depiction of HAdV-C2/5 entry into epithelial cells. For infection of polarized epithelial cells, which lack HAdV-C2/5 receptors on the luminal apical side, alternatively spliced forms of Coxsackie virus B AdV receptor (CAR) can be transported apically in low amounts.255 In addition, innate immune responses of macrophages and other immune cells stimulate the expression of cytokines and chemokines, such as IL-8, and thereby lead to apical localization of CAR and integrin receptors.55,56 Interleukin-8 binds to the CXCR1/2 receptor and triggers translocation of CAR and integrin receptors to the apical surface of epithelial cells. (1) This allows virus attachment to CAR exposed on the cell surface, followed by drifting motions and retrograde flow mediated by F-actin and myosin-2 whereas particles are still outside the cell.34,256 Eventually, virus binds to integrins, which are immobile and have a lower affinity to the virus than CAR.63,257 (2) Mechanical forces generated by receptor dynamics initiate the stepwise uncoating process.172 Fiber shedding and the release of limited amounts of the membrane lytic protein VI from the virus occur on the cell surface.34,35,258 This is likely aided by interaction between integrins and penton base, inducing a clockwise untwisting of the pentamer and thereby loosening the viral capsid.144 (3) Transient protein VI-mediated permeabilization of the plasma membrane and Ca2+ influx elicit lysosomal exocytosis, a cellular response to membrane injury.40 Secreted lysosomal acid sphingomyelinase (ASMase) generates ceramides. (4) Virus endocytosis requires integrin signaling and is clathrin-mediated and dynamin-dependent. (5) As virus is endocytosed, additional protein VI is released and interacts with ceramide lipids to enhance endosomal breakage. Thereby, the virus is released to the cytosol and avoids passage through acidic compartments.40,173 (6) Partially uncoated HAdV-C2/5 binds to cytosolic dynein via hexon and travels on microtubules toward the microtubule organizing center.21,225,226,259 (7) Virions dock on the cytosolic side of the nuclear pore complex (NPC) by engaging with the nucleoporin Nup214.232,243,244,246 Simultaneously, protein XI and Nup358 bind to kinesin-1, which exerts forces on the capsid and the NPC and thereby sets free the viral DNA genome and displaces a fraction of the nucleoporins.246 (8) Viral DNA in association with the condensing protein VII is imported into the nucleus.229,232,260,261

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hypervariable regions (HVRs), which are subject to immune recognition by both neutralizing antibodies and virus-specific T-cell responses.17 They also interact with host proteins, including blood factors and cytoplasmic motors.18–21 A trimeric fiber is anchored at each vertex to a pentameric penton base, which binds to integrins and controls virus uptake and cell signaling (for reviews, see Refs 13, 22, 23). The capsid is held together with a range of cementing proteins, as indicated by an X-ray crystal structure and a cryo-electromagnetic (EM) structure.24,25 Neither the function nor the location of these cementing proteins is entirely clear, however. In particular, the cryo-EM and the X-ray structures of AdV are not in complete agreement with respect to the location of the cementing proteins. For example, the cryo-EM structure localizes protein IIIa beneath the vertices, in close association with protein VIII,24 whereas the X-ray structure assigns protein IIIa to a four-helix bundle on the outer surface of the coat, aligned along the icosahedral edges.25 The X-ray structure places the inner cementing proteins VI, VIII, and the deoxyribonucleic acid (DNA) core protein V near the vertex, and protein IX outside the virion. The vertex anchors the fiber protein, which protrudes away from the capsid and attaches virus to cell surface proteins or sugars (Table 1).26,27 The capsid harbors a single copy of a linear double-stranded viral DNA. The capsid is a metastable structure that progressively uncoats in response to cellular cues. This feature ensures diverse functions of the capsid, such as endosomal escape, DNA shielding during cytoplasmic transport, and finally, DNA release for nuclear import (for a review, see Ref. 16). Meta-stability of the capsid is a key feature and is largely controlled by the viral cysteine protease, L3-p23 (AdV protease, AVP), which cleaves the precursors of protein IIIa, VI, VII, VIII, X, terminal protein, and the scaffold protein L1-52/55K (reviewed in Ref. 28). L1-52/55K is important for the assembly of the particle because it provides a bridge between the inner wall of the coat and the viral DNA. Its cleavage by AVP spring-loads the particle to a metastable state capable of responding to cellular cues for uncoating.29,30 Uncoating is the process by which the viral genome is exposed and then separated from the protein or lipid coat. For example, the temperature-sensitive mutant HAdV-C2_TS1 (TS1) has a packaging-defective AVP and fails to open the coat and release the DNA. Interestingly, the surface of TS1 is indistinguishable from that of wild-type HAdV-C2, although TS1 has more mass in the vertex region than HAdV-C2.31,32 Because the vertex is the weakest part of the icosahedral coat,30,33 it is possible that limited proteolysis of vertex proteins renders the particle metastable. It is likely that TS1 cannot interpret the mechanical cues it receives from the differential movements of the viral receptors on the cell surface (described in Fig. 1), unlike HAdV-C2, which is responsive to these cues and exposes the membrane lytic protein VI.34–36 This is compatible with the notion that TS1 binds to cells and is endocytosed, but fails to uncoat and does not deliver its genome to the nucleus.34,37–40

3.  Attachment Factors and Signaling Receptors To identify a target cell, AdVs rely on their affinity for attachment factors and signaling receptors. Some receptors are readily available on the site of entry whereas others such as Coxsackie virus B AdV receptor (CAR), desmoglein-2 (DSG2),

Host Factors Binding to Adenoviruses, and Their Endocytic Uptake Pathways Expression

Exposure

Serotype

Affinity

CAR

Broad

Tight junctions; low apical abundance of CARex8

2,5,9,12 41L64,262

CD46

Broad

DSG2 SA

Broad Broad

Apical and basolateral264 Desmosome Cell surface

2,4,5,9,12, 15,19,31,41,52 and others, except species B,D and G101,142,262 3,7,11,14,16, 21,35,49,50265 3,7,11,14 8,19,37,52

HSPG

Broad

Cell surface, ECM

2,5113 3266

Integrins

Broad

Basolateral surface

2,9136,143

SR

Macrophages, other cell types114,268 Blood

Cell surface

Most types, except 40,41, CAdV-2 518,118

2,3,5,6,7,11, 13,16,18,35, 37,46,49,50; except 20,29,25,17, 26,28,44,48a

5153

FX

Soluble

3,7,11,14, 16,21,35 391 37,5299,101

Avidity

Virus Binding Domain

Pathway

Fiber knob

Clathrin–dynamin; lipid rafts mediated for HAdV-C5 or fiber knob of canine Ad2 in neurons263

3,777

Fiber knob

Macropinocytosis81,82

390 37103

Fiber knob Fiber knob, interactions of +/– charges KKTK motive in fiber shaft Penton base, fiber knob111,266 RGD domain in penton

? Caveolin for HAdV-D37 in corneal cells204 ?

Hexon hypervariable regions (HVRs) 1, 2, 5, 7 T423, E424, T425 of HVR7 and K10 in FX GLA domain19

Macropinocytosis?

3

2,5257,267

5,35153

Clathrin–dynamin; macropinocytosis38

Residues R93, K96, R125, R165, K169, K236, and R240 in SP domain of FX bind to HSPG.150 Endocytic pathway?

Adenoviral Vectors for Gene Therapy

Binding Factor

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Table 1 

Body fluids

Soluble

1,2,5,6

DPPC

Lung surfactant

Soluble or intracellular secretory organelles270

2,5

α-Defensins HD5, HNP1

Extracellular Soluble fluids, nasal, lung, female urogenital tract epithelia146 Blood, Soluble extracellular fluids

Secreted Ig

2,3,5,7,11, 12,14,16,35 4,19,23,25 37,41,51

Most serotypes, including MAdV-1

2

5271,272

IIIa and hexon: CAR-independent160,165 Far-Western blots of soluble biotinylated-­bovine Lf269 Hexon: CAR-independent167 Flotation assay and sodium dodecyl sulfate–polyacrylamide gel electrophoresis269 Between penton base CAR-mediated and fiber271 uptake, inhibition of uncoating Enhanced infection271

Variable

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Lf

Virus neutralization by C3b coating; Ig-mediated phagocytosis154,273,274

Cell-bound and soluble factors binding to adenoviruses are depicted in light gray and dark gray, respectively. Affinities were derived from binding strengths of soluble virus factors (trimeric fiber knob, penton base, hexon) to host receptors. Avidity represents synergistic but not additive interactions of receptors or cells with highly repetitive ligands such as viruses, penton dodecahedrons (PtDd), oligomeric viral proteins. High affinity or avidity are defined here as KD < 1 μM, indicated in bold. Information about expression of CAR, DSG2, and CD46 can be found at: http://www.proteinatlas.org/ENSG00000117335-CD46/tissue. http://atlasgeneticsoncology.org/Genes/GC_DSG2.html. http://www.proteinatlas.org/ENSG00000154639-CXADR/tissue. 41-L, long fiber of HAdV-F41; 52-S, short fiber of HAdV-G52; Coxsackie virus B AdV receptor (CAR). aK is indicated for HAdV-C5, HAdV-B35, and HAdV-D26. The other serotypes are grouped together if they have similar binding properties. D

31

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or integrins, are scarce or constrained to the basolateral surface of polarized epithelia. Besides attachment, CAR, integrins, CD46, or DSG2 themselves have a crucial role in virus endocytosis, uncoating, and signaling. These aspects determine productive entry, gene expression, and infection. For most of the other factors proposed to mediate AdV attachment to cells, a specific role in infection has not been identified. Hence, their subsequent discussion will be descriptive.

3.1  Accessing the CAR The CAR is an immunoglobulin family protein implicated in cell adhesion, migration, and growth. It is highly expressed in the myocardium, intestines, pancreas, lung, liver, and kidney and is present in low amounts in adult brain or cancer cells.27,41,42 CAR connects neighboring cells through homophilic contacts at the lateral and basal surfaces of polarized cells.43,44 It is not readily exposed to viruses that approach the apical side of polarized epithelia. Nonetheless, it is a high-affinity attachment factor for most HAdVs with the exception of the B, D, and G types.45–50 It also binds to canine AdV-2 (CAdV-2) and avian AdV CELO.51 Three mechanisms have been implicated in rendering CAR accessible to HAdVs and enabling infection of the respiratory, gastrointestinal, and ocular tracts. The first is disruption of tight junctions by a preexisting condition such as physical or chemical stress in asthma, cystic fibrosis, and chronic obstructive pulmonary disease.52–54 Second, HAdV-C2/5 subverts the innate immune response from macrophages to increase apical CAR availability.55,56 HAdV-C2/5 is taken up by alveolar macrophages and triggers the release of proinflammatory chemokines and cytokines. Among these, interleukin 8 (IL-8 or cysteine-X [any amino acid]–cysteine type chemokine receptor [CXCL-8]) induces the translocation of CAR and integrin co-receptors to the apical surface of polarized epithelia.55 CAR relocalization to the apical membrane depends on CXCR-1/2 and Src tyrosine kinases, and mimics a cell migration phenotype. The third mechanism occurs when IL-8 enhances the expression of an alternatively spliced CAR isoform, CARex8, which traffics in low amounts to the apical surface owing to its C-terminal tail.56,57 CARex8 expression is associated with activation of protein kinase B (or AKT) and S6-kinase and inhibition of glycogen synthase kinase-3β. Virus binding to CAR enhances signaling through p42/44 extracellular kinase-1,2 and increases expression of the entry receptors β1/3 integrins.58,59 Signaling cascades are reinforced during viral gene expression, when the viral immediate early protein E1A stabilizes the messenger RNAs encoding for proinflammatory cytokines, including IL-8.60 That CAR has a role in inflammatory processes is supported by the observation that in an inflamed mucosal tissue, this receptor is liberated from its homophilic contacts on polarized cells and engages in activation of immune cells such as neutrophils or gamma-delta T cells.56,61 In addition to a well-characterized function in entry, CAR has a role in virus egress from infected epithelia. At late stages of infection when viruses are assembled in the nucleus, structural viral proteins are produced in large amounts, and some of them, including fibers and penton base, exit the infected cell by unconventional secretion.43,62 The released fibers bind to the lateral surface of CAR, akin to homophilic CAR-CAR

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binding at basolateral cell adhesion sites of polarized cells.63 This binding dissolves cell adhesions, most likely involving avidity effects owing to the trimeric nature of the fiber knob.64 This shows that CAR is a versatile entry and egress receptor for a large number of mastadenoviruses, including human, simian, canine, and chicken AdVs.

3.2  Using CD46 in High- and Low-Affinity Modes Many types of the species B and D, HAdVs attach to the membrane cofactor protein CD46 (see Refs 13, 26). CD46 is an inhibitor of complement activation and is present on all nucleated cells.65 It is localized apically in polarized epithelial lung tissues,66 although it can also be expressed basolaterally in other tissue types.67,68 Virus binding to CD46 occurs top-down on tilted CD46, which suggests that straight fiber shafts projecting from the virus are suitable for this interaction. The trimeric fiber knob attaches to a short consensus repeat in the extracellular variable loop of CD46.69–76 The knobs of HAdV species B have evolved affinities to CD46 ranging from low nanomolar for species B2, such as HAdV-B11 or B35, to several micromolar for species B1, such as HAdV-B3 or B7. The low apparent affinities are compensated for by avidity mechanisms, which allow the B1 HAdV to bind to CD46-expressing cells with nanomolar affinity.77 We term this “low-affinity, high-avidity binding.” This type of binding is possible for receptors that are highly expressed. CD46 protects from homologous complement-mediated cell killing, maintains the homeostasis of epithelial barriers, and promotes formation of cellular cytoskeleton.78,79 Human adenovirus attachment to CD46 triggers receptor clustering and macropinocytosis.80–82 Ligands of CD46, including fiber knobs of HAdV-B35 and HAdV-B11p, rapidly downregulate surface expression of CD46.83–85 Together with degradation of CD46, this enhances complement-mediated lysis, alters antigen presentation on major histocompatibility complex (MHC) I and II, and decreases IL-12 secretion in activated human peripheral blood mononuclear cells.86–88 In summary, CD46 is a versatile receptor for both species B1, which preferentially infect respiratory epithelia, and B2 HAdVs, which predominantly infect the kidney and urinary tracts. CD46 captures HAdV-B and -D with a range of affinities and may function as a docking site for viruses on the apical domain of polarized cells. It will be interesting to determine whether highly pathogenic HAdV-B such as B14 also use CD46. Together, AdVs exploit two different strategies for cell attachment. The first involves highaffinity receptors and ensures binding even when only scarce amounts of receptors are available. The second strategy relies on high avidity toward abundant but lowaffinity attachment sites and may provide opportunities for expanding viral tropism and pathogenicity.

3.3  Desmoglein-2 The species B1 HAdVs not only bind to CD46 but also exploit another receptor, DSG2.77,89–91 The interaction between DSG2 and HAdVs is likely of low affinity and is enhanced by avidity. This is suggested by the observation that only intact virus or penton dodecahedron (PtDd) complexes composed of penton base and fibers, but

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Adenoviral Vectors for Gene Therapy

not trimeric fiber knob, measurably attach to DSG2. DSG2 participates in the formation of desmosomes and is largely restricted to the basolateral side of polarized epithelia.92 The role of DSG2 in a primary infection in vivo remains unclear, in part because mouse DSG does not bind to HAdV-B3.89 It is speculated that HAdVs might exploit DSG2 for virus spreading, because cells infected with species B or D viruses release high amounts of PtDd before cell lysis. These assemblies consist of fibers and penton bases,93,94 interact with DSG2, and initiate a signaling cascade that mimics epithelial to mesenchymal transition.90 Epithelial to mesenchymal transition widens cellular junctions and exposes basolateral factors, thereby facilitating lateral spreading of viral progeny.93 In summary, whereas CD46 is a unique receptor for HAdV-B16, HAdV-B21, HAdV-B35, and HAdV-B50,89,95 HAdV-B3, and HAdV-B7 bind to DSG2 and engage with CD46 in an avidity-enhanced mechanism.77 HAdV-B14 and B11 use both receptors with comparable affinities.95 It is conceivable that DSG2 and CD46 are independent attachment sites for HAdV-B types: that is, DSG2 at desmosomal junctions and CD46 on apical and basolateral surfaces.66,96,97 It is possible that apical CD46 leads to activation or degradation of DSG2, and thereby opens cell–cell contact sites for viruses to access the basolateral pool of receptors, including DSG2 and integrins. Surprisingly, despite the nearly undetectable apical binding, HAdV-B35 efficiently transduced polarized Caco-2 from the apical side.98 It will be interesting to determine the underlying mechanisms of this process.

3.4  Sialic Acid Sialic acid (SA) and its variants are low-affinity attachment sites (dissociation constant (KD) between 19 and 37 μM) for epidemic keratinoconjunctivitis causing HAdV-D8, HAdV-D19, and HAdV-D37, and the gastroenteric HAdV-G52.99–101 Sialic acid is highly abundant on the apical side of polarized epithelial tissues, and might therefore serve as an initial anchor for primary infections despite the low binding affinity. The family of sialic acid encompasses over 50 related sugars with variable chemical modifications, linkages, and tissue expression. HAdVs exploit the ubiquitous Neu5Ac, which is similar to the glycans of GD1a gangliosides.102 HAdV-D8, HAdV-D19, and HAdV-D37 harbor three conserved positively charged binding sites (pKa = 9) on the fiber knob that interact with the negatively charged Neu5Ac (pKa = 2.6).103,104 HAdV-G52 contains a long and a short fiber. Although the long fiber binds to CAR, attachment to some cells also depends on the short fiber, which interacts with sialic acid.101 Sialic acid is also highly abundant on erythrocytes and may be involved in AdV-mediated hemagglutination and in determining the biodistribution of HAdVs and their vectors.105

3.5  Heparan Sulfate Heparan sulfate–containing proteoglycans (HSPG) are linear chains of polymerized disaccharides (approximately 80 residues) attached to either the extracellular matrix

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(ECM) or cell surface components, where they serve to reinforce cell adhesion.106 Interaction with fibronectin regulates the formation of focal adhesions and stress fibers.107 HSPG are involved in a multitude of cellular processes, including blood coagulation108 and liver lipid metabolism.109 They act as attachment sites for HAdVs that are free or coated with other soluble factors (see also Section 3.8). They are reported to have low affinity for HAdV species C serotypes, and possibly HAdV-B3 and HAdV-B35, which may facilitate downstream interaction of the viruses with integrins.110–112 Whereas binding of HAdV-C5 occurs through a positively charged lysine– lysine–tyrosine–lysine (KKTK) motif in the fiber shaft,113 HAdV-B3 apparently uses fiber knob.111 Mechanisms of HAdV-B35 binding remain unclear and physiological consequences are difficult to elucidate.

3.6  Scavenger Receptors and Other Attachment Factors Scavenger receptors (SR) encompass eight classes of proteins with low sequence homology. They have the propensity to bind negatively charged molecules, are highly expressed on macrophages, and expose an electrostatic positive patch implicated in ligand binding.114,115 SR have been implicated in the clearance of HAdV vectors by Kupffer cells in the liver.116–118 Kupffer cells are considered detrimental to systemic adenoviral therapies because they clear a large fraction of the injected HAdV-C5– based vectors.119–121 They channel the vector to a degradation pathway, which leads to necrosis and inflammation.122,123 Interestingly, HAdV-C6 or chemically shielded HAdV vectors evade clearance by Kupffer cells.117,124 The scavenger receptor A-II (SR-A) mediates macrophage uptake of HAdV-C5– based vectors, as shown, for example, by gain-of-function experiments.18,125–127 This occurs through external negatively charged HVRs 1, 2, 5, and 7 of hexon, which interact with SR-A. In addition, respiratory tract infections with HAdV are likely to involve alveolar macrophages, which tune the cytokine profiles such that epithelial cells expose HAdV receptors CAR and integrins (see Section 3.1).55 Further investigations are required to elucidate the physiological roles of SR and other attachment factors in natural and therapeutic infections of model organisms and humans. It remains to be seen whether such factors include the previously proposed candidate receptors CD80/CD86 and MHC I.128–131

3.7  Internalization Co-receptors: Integrins Integrins are internalization and signaling co-receptors for most HAdVs, except, for example, HAdV-F40 and HAdV-F41, which lack an arginine-glycine-aspartate (RGD) motive in penton base.13,26 Integrins anchor cells to the ECM, transduce signals to and from the cell, and regulate cell survival, migration, and differentiation.132 They are located on the basolateral side of polarized epithelia but can be relocated apically in the context of infection. Integrins are in a dynamic equilibrium between active ligand-bound and inactive ligand-free states. Distinct integrin conformations are regulated by binding of extracellular divalent cations (outside-in) and cytosolic proteins (inside-out).133

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Adenoviral Vectors for Gene Therapy

Interaction of integrins with the endocytic machinery critically determines integrin turnover.134 Integrins occur as 24 α/β heterodimers of 18 α and 8 β subunits. Interaction between HAdV, αvβ3, and αvβ5 are best characterized, but HAdVs have also been shown to engage with αvβ1, αMβ2, and α3β1.135–140 Human AdV binding to integrins requires an RGD sequence in penton base and mimics attachment of integrins to RGD-containing ECM proteins such as laminin, fibronectin, and vitronectin.141 HAdV-C2 internalization, but not binding to CAR-positive cells, was inhibited by soluble penton base or synthetic RGD peptides.136 Nevertheless, in the absence of CAR, integrins can serve as attachment receptors.139,142 Remarkably, HAdV-D9 pentameric penton base and αVβ3 interact with a KD in the nanomolar range, and one penton base of HAdV-A12 engages with a maximum of four αvβ5s.143,144

3.8  Extracellular Factors Influencing Viral Tropism Body fluids contain factors that bind to AdVs, confer alternative viral tropism, and influence the outcome of both natural infection and systemic gene therapy. For example, dipalmitoylphosphatidylcholine (DPPC) in lung surfactant or lactoferrin (Lf) in secretory fluids apparently enhance infection of polarized cells regardless of CAR availability. A range of blood coagulation factors, such as factor X (FX), FVII, FIX, and protein C binds to HAdV-C5 and its derived vectors.145 For example, FX shields circulating viruses from immunoglobulin (Ig) M and complement-mediated neutralization and influences virus liver tropism. Extracellular released defensins inhibited uncoating and delayed infection with some serotypes and enhanced infection with others, such as HAdV-D and F (Table 1).146 This suggests that defensins modulate attachment of HAdV to the cell surface.

3.8.1  Coagulation Factors Vector bioavailability in systemic HAdV therapies is restricted by rapid neutralization through Kupffer cells or intrinsic viral liver tropism. A soluble ligand suspected to orchestrate liver targeting in mice and nonhuman primates is blood coagulation FX.147–149 Factor X is a vitamin K–dependent coagulation factor that circulates in the bloodstream, attaches to intravenously injected HAdV-C5–based vectors, and bridges them to HSPG via the FX γ-carboxyl-glutamic acid–rich (GLA) domain.150 Cryo-EM experiments identified the solvent-exposed hexon HVR7, particularly amino acids T423-E424-T425 and E451, as essential for binding to FX and hepatocyte transduction.19,151,152 Not all HAdV species bind to FX and binding affinities depend on the amino acid sequence of HVRs. For example, HAdV-C5 and HAdV-C2 are strong binders (for HAdV-C5, the KD is about 2 nM), whereas HAdV-B3 and HAdV-B35 are weak binders.153 Although several reports have pinpointed that FX is detrimental for AdV-based therapies, it has emerged that FX is also beneficial because it shields HAdV from rapid neutralization by IgM and the complement system.154 For further reading on coagulation factors and liver targeting, see, for example, Lopez-Gordo et al.155

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3.8.2  Immunoglobulins and Complement Ablation of FX binding to HAdV-C5 by the vitamin K inhibitor warfarin renders capsids accessible to circulating IgM and the complement components C1q and C4.154 These factors not only trigger an antiviral cytokine response but also directly coat viruses to prevent capsid binding to target cells, including hepatocytes.154,156,157 Accordingly, high levels of HAdV-specific IgM efficiently blocked liver transduction in mice.127 Intriguingly, the endosomal escape-defective mutant TS1 binds to antibodies and elicits early complement cascade activation in vitro but not in vivo.158 These data suggest complex dynamics of complement activation in vivo. Complement activation is influenced by cell damage signals or other events associated with infection. Immunoglobulin coating of viruses also confers tropism to specialized immune cells that internalize viruses by phagocytosis11 (see also Section 4.3).

3.8.3   Lactoferrin Lf is a globular glycoprotein present in mucosa and body fluids such as tears, nasal fluids, and saliva.159 Using tear fluids, it was found that Lf enhances infection of cultured human cells with HAdV-C5 but not the eye disease associated HAdV-D37.160 It was suggested that Lf acts as a bridging factor between HAdV and the cell. An Lf-mediated increase in HAdV transduction was apparently independent of CAR. At high concentrations, however, Lf inhibited HAdV infection. This suggested that Lf competed with the HAdV–Lf complex for binding to an as-yet unidentified receptor.161,162 A candidate receptor for the HAdV–Lf complexes is the lactoferrin receptor (LfR), which is expressed on human epithelial cells and T cells.163,164 Another receptor candidate is the lectin DC-SIGN, which recognizes the mannose-type N-linked glycans on Lf and triggers transduction of monocytes and dendritic cells by HAdV5-Lf.165,166 It will be interesting to determine how Lf binds the viruses and enhances or inhibits infection of CAR-negative immune cells or polarized cells.

3.8.4   Dipalmitoylphosphatidylcholine Major macromolecules in alveolar surfactant are lipids, proteins, and polysaccharides. One of the lipid species, disaturated DPPC, was found to enhance infection of cultured lung epithelial A549 cells with HAdV-C2, as judged by transgene expression.167,168 Purified viruses or hexons are cofractionated with liposomes. The authors interpreted the data to mean that DPPC would enhance HAdV-C2 infection of cells in the absence of virus receptors, and perhaps gate virus entry from the apical side of polarized epithelial cells. Mechanistic data will be required to further support this idea.

4.  Endocytosis HAdVs enter cells by endocytosis, a vital process that occurs in all cells. Endocytosis is the uptake process for fluids, solutes, membrane-associated proteins, and membranes

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as well as receptor ligands. It determines cell fate and well-being, including the engulfment of nutrients, sensing of the environment, and tuning of signaling.169–171 Endocytosis occurs through the clathrin-mediated pathway, macropinocytosis, phagocytosis, caveolae-dependent uptake, and the CLIC/GEEC, IL-2, Arf6-dependent and flotillin-dependent pathways. These pathways require subsets of effector molecules and are subject to distinct regulations. AdVs evolved to exploit multiple pathways of internalization. Endocytic uptake of HAdV-C2/5 is highly effective in receptor-positive epithelial cells, where > 80% of bound viruses are endocytosed with the half-time of 5–10 min.38,81,82,172,173 This is a great advantage for the virus because rapid endocytosis clears viral traces from the cell surface and limits the risk of premature detection of the infection by the immune system.

4.1  Clathrin-Mediated Endocytosis Clathrin-mediated endocytosis (CME) is initiated at specialized membrane domains rich in phosphatidyl-inositol-4,5 bisphosphate (PI4,5P2). There, the endocytic cargo is recognized by cytosolic adapters that recruit adapter proteins, clathrin, and other factors for vesicle formation and scission.174,175 Human AdV-C2 and HAdV-C5 gain access into epithelial cells by clathrin- and dynamin-mediated endocytosis. Initial EM studies revealed a clathrin coat on endocytic pits and vesicles enclosing incoming HAdV.176–179 Further studies with HAdV-C2/5 employing dominant-negative mutants revealed the requirement of clathrin, dynamin, and Eps15 for endocytosis and infection.38,180,181 The endocytic process of HAdV is enhanced by β-integrins.135,136,182 Many β-integrins harbor an asparagine-proline-any amino acid-tyrosine (NPXY) or NPXY-like motive in their cytoplasmic domain, which binds to cargo selectors such as NUMB and Dab2. NUMB directly interacts with the adapter AP-2 and clathrin. The site of interaction surrounds the focal contacts, where NUMB is involved in integrin trafficking during cell migration.183 Dab2 is another adapter molecule that initiates CME by bridging β-integrins with clathrin and PI4,5P2. Interestingly, Dab2 supports CME in AP-2–deficient cells.184,185 This suggests a certain redundancy, perhaps ensuring efficient endocytosis under diverse conditions. Membrane shaping might require actin, depending on the cell type. Actin is not required for endocytosis in nonpolarized cells, yet in polarized cells it has been implicated in the invagination of membrane domains on the apical but not the basolateral side. Actin is thought to provide a force to counteract membrane tension and complete membrane bending and pit constriction.186 F-Actin and the clathrin lattice are connected by huntingtin interacting protein 1 related (HIP1R).186 During HAdV entry, actin dynamics are tuned by signaling through integrins involving PI3K, p130CAS, and the actin remodeling GTPases Rac1 and Cdc42.135,187 The clathrin-coated pits are finally subjected to closure and scission by dynamin 2 (Dyn2) in epithelial and immune cells. The expression of Dyn2_K44A, a dominant negative form of Dyn2 defective in GTP binding, blocks HAdV-C2/5 at the cell surface. Supported with RNA interference of Dyn2, these data imply that the major pathway of HAdV-C2/5 entry into epithelial cells is dynamin dependent.38,180

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4.2   Macropinocytosis Macropinocytosis is the engulfment and uptake process of large amounts of fluids and membranes. It occurs by actin-dependent membrane protrusion and retraction, and results in large intracellular vacuoles (>0.2 μM), referred to as macropinosomes, which can mature to late endosomes.188 Constitutive macropinocytosis contributes to antigen presentation in macrophages and dendritic cells, and is widely exploited by pathogens to modulate the immune response.11,189 HAdV-B3 and HAdV-B35 subvert macropinocytosis for infection of hematopoietic and nonpolarized epithelial cells.81,82 They cluster CD46 and αV integrins and activate cytosolic Rac1, a small GTPase coordinating actin remodeling and plasma membrane blebbing. Downstream of Rac1, p21-activated kinase (Pak1) activates the C-terminal binding protein 1 of E1A (CtBP1).190 CtBP1 and Pak1 colocalize with macropinosomes that contain the fluid phase-marker dextran, viruses, CD46, and αV integrins.81 Remarkably, activation of CtBP1 might influence infection beyond membrane trafficking. CtBP1 is a transcriptional regulator and possibly inhibits the AdV transactivator E1A during viral gene expression.191,192 Incoming HAdV-B that use CD46 have been shown to activate TLR9.193 This could result in the upregulation of CtBP1-repressed genes and thereby promote an antiviral state in the infected cell.194–196 This scenario suggests that CtBP1-dependent macropinocytosis is a defense reaction against HAdV. In support of this hypothesis, HAdV-C2/5 induce macropinocytosis, although they do not engage with CD46. For these viruses, macropinocytosis is not the infectious pathway and is slightly delayed compared with their main uptake pathway, CME.180 All of these observations highlight the broad significance of macropinocytosis for infection, ranging from a virus uptake pathway to stimulating innate immunity.

4.3   Phagocytosis Phagocytosis mostly accounts for pathogen neutralization and antigen presentation: for example, when specialized immune cells internalize opsonized viruses.11,197 Immunoglobulins contain constant (Fc) and variable domains, coat HAdV, and trigger phagocytosis after attaching to Fc receptors (FcR) on antigen-presenting cells, macrophages, and dendritic cells.198 For example, IgG-clustered HAdV-C5 particles are internalized by dendritic cells via the FcγR2 and FcγR3 receptors.199 Human AdVs exploit the Fc-mediated pathway for infection. An example is the uptake mechanism of HAdV-C2-CARexFc clusters. CARexFc is a fusion protein carrying the extracellular domain of CAR and the constant region of a human IgG.200,201 The clusters, which are several micrometers in diameter, transduced CAR-negative hematopoietic cells by phagocytosis through the Fc receptor FcγR1 (CD64). Intriguingly, other studies found that HAdVs in complex with IgG, IgM, or IgA were delivered to the cytosol and elicited a virus-neutralizing innate immune response triggered by the cytosolic FcR TRIM21.198,202,203 This highlights that a cytosolic antibodymediated barrier defends the host against incoming viruses.

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4.4  Caveolar Endocytosis It has been reported that the keratoconjunctivitis-causing HAdV-D37 enters corneal cells by caveolar endocytosis.204 Corneal cells have high levels of constitutive endocytosis and form multiple flask-shaped pits lacking a clathrin coat, classified as caveolin-containing pits. Interestingly, corneal cells of caveolin knockout mice are less susceptible to HAdV-D37 entry and HAdV-D37-dependent activation of Src kinase and CXCL-1 production. It remains to be shown whether caveolar uptake controls HAdV-D37 infection or whether caveolae function as a signaling hub in controlling HAdV-D37 infection.

5.  Endosomal Escape HAdVs penetrate cellular membranes from endosomes. Upon initial steps of uncoating and endocytosis, the inner capsid protein VI is externalized and disrupts the limiting membrane. This strategy avoids detrimental lysis of the plasma membrane, limits the damage to intracellular endosomes, and ensures clearance of viral traces from the cell surface.

5.1  Protein VI for Membrane Lysis Endosomal escape is efficient, with a success rate of 80–95% for HAdV-C581,173,205 and occurs rapidly. Human AdV-C2 and HAdV-B3 are found in the cytosol within 15 min after cell attachment.173 Endosomal penetration of HAdV is coupled to the stepwise nature of uncoating, as indicated by studies of HAdV-C2/5 and the mutant TS1. The TS1 mutant is not infectious. It remains trapped in endosomes, and is degraded in lysosomes.206 Underlying reasons are an uncoating defect of TS1, which precludes exposure of the membrane lytic factor VI. This defect occurs because TS1 fails to respond to mechanical cues from receptors on the plasma membrane. Mechanical forces occur when viruses bind to CAR and undergo acto-myosin–dependent drifts that are counteracted by immobile integrins. These events trigger fiber release followed by externalization of VI from a location within the coat.24,144,207,208 TS1 is further defective at releasing the tight interactions between the DNA core and the inner side of the coat.30–32,34,275 Electron microscopy analysis revealed that wild-type AdVs are largely intact when they reach the cytosol,209 which suggests that endosomes are lysed rather than perforated with discrete pores. In support of this notion, a decrease in total endosomes after infection was measured.210 It was also observed that HAdVs enhance cytosolic or nuclear co-delivery of macromolecules, such as 70-kDa fluorescein isothiocyanate dextrans or bovine serum albumin coupled to a nuclear localization peptide.180,211,212 In addition, the ribotoxin α-sarcin was found to intoxicate cells in a vATPase–dependent manner when added to cells together with HAdV-C5.36,213 Any co-delivery assay is an indirect measurement of virus penetration because it measures leakage of any endocytic organelle formed during virus and toxin co-uptake. In contrast to co-delivery

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assays, virus penetration can be addressed directly at the single-cell, single-virus level. Direct measurement of virus penetration combined with infection studies has shown that HAdV-C2/5 and HAdV-B3 penetrate endosomal membranes independently of the vacuolar endosomal H+-ATPase inhibitor bafilomycin A1 or the protonophore niclosamide.173,210,214,215 Remarkably, however, virus penetration was sensitive to lysosomotropic agents, which suggests that the ionic milieu beyond protons is important for HAdV penetration.172,173 Membrane lysis requires the activity of the viral membrane lytic factor protein VI.216 In vitro studies revealed a role of VI in lysis of model membranes such as liposomes, notably independent of low pH.36 Liposomes exposed to VI collapsed and formed tubular structures. Fragmentation is enhanced in the presence of a lipid that induces positive curvature, lyso-PC.205 Membrane disruption depends on the N-terminal 54 amino acids of VI.36 Particularly, amino acids 36–53 of VI likely fold into an alpha helical structure with amphipathic properties that allow association of VI in parallel to the lipid bilayer, as suggested by tryptophan depth profiling experiments.217 That the amphipathic N-terminal domain is important for virus penetration was shown by virus mutagenesis experiments. The mutation of alanine to glutamine at position 40 (VI-L40Q) reduced not only the membrane lytic activity in vitro but also cell infection.216 Another mutation in the alpha helical domain, a glycine at position 48 (G48C), resulted in aberrant disulfide bonds and the formation of VI dimers, and decreased membrane lysis.218 Intriguingly, the N-terminal amino acids 34–50 of protein VI do not form a secondary structure within the intact capsid.18 It is conceivable that in endosomes, protein VI encounters cellular cues, such as lipids or specific ions, that favor folding of its lytic domain into a membrane active amphipathic alpha helix. The lytic mechanism might resemble that of antimicrobial amphipathic alpha helical peptides,219 some of which insert their hydrophobic domain into the hydrocarbon backbone of membranes and traverse the lipid bilayer.220

5.2  A New Concept: Hijacking Membrane Repair for Endosome Lysis Endosomal lysis is not thought to be a physiological event. However, although membrane penetration strongly depends on viral proteins, host factors contribute in multiple ways to this process. For example, cues from the receptors and mechanical forces are indispensable for VI exposure.34 In addition, new data indicate that incoming viruses release few copies of VI at the cell surface, and thereby induce transient membrane permeabilization.40 This triggers the influx of calcium ions and lysosomal exocytosis, a plasma membrane repair response associated with the increase of ceramide lipids. Ceramide lipids enhance virus endocytosis and interact with protein VI in endosomes, thereby enhancing endosomal lysis. This provides evidence for a positive feedback loop between limited virus uncoating, protein VI-mediated membrane lysis, and localized lipid production. It is likely that besides ceramide, other endosomal lipids and host factors influence virus uncoating and membrane stability, such as ion fluxes, cytosolic factors, or lipid and protein modulating enzymes.205,221

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6.  Targeting the Nucleus HAdVs engage with cytoskeleton-dependent motor proteins to transit the cytoplasm from the site of endosomal escape to the nucleus.

6.1  Transport through the Cytoplasm Long-range cytoplasmic transport is largely mediated by microtubules (MT) and associated motors. Microtubules are polarized filaments composed of α−β tubulin heterodimers. In nonpolarized cells they nucleate at the microtubule organization center (MTOC) near the nuclear periphery.222 In polarized epithelial cells, microtubules nucleate at the apical side and elongate toward the basal site, where the positive end is situated.223 Motor proteins carry cargos across cells along microtubules. For this, motors engage in either minus end–directed motions toward the MTOC (dynein), or plus end–directed motions toward the periphery (kinesin).224 HAdVs have been shown to traffic in bidirectional modes on MT.225–228 Adenoviruses exploit the MT motor complex dynein–dynactin to reach the nucleus of epithelial cells. They reach the nucleus as partially dismantled DNA-positive capsids 30–60 min after entry.225,227–232 HAdV binding to the dynein motor likely occurs through hexon, as supported by stochastic simulation modeling of virus motions on microtubules.233 In addition, low pH-primed hexons directly interact with the light intermediate chain 1 and the 74-kDa intermediate chain of dynein, presumably through the HVR1 loop.21,234 HVR1 is susceptible to cleavage by the bacterial protease dispase, and this cleavage abrogated hexon binding to dynein in vitro. However, HAdV-B/C does not pass through acidic compartments and is insensitive to inhibitors of endosomal acidification.38,173,205 Cues other than low pH are therefore likely to tune the conformation of hexon and promote binding to dynein–dynactin. Dynein-dependent motions are subject to regulation. The dynein cofactor dynactin regulates motor processivity and enhances HAdV motions.225,226,235 In addition, dynein-dependent motions are enhanced by virus signaling from the cell surface, for example, through the cAMP-dependent protein kinase A (PKA), the Rho family GTPases RhoA and Rac1, and MAP kinase p38.59,236,237 It is possible that PKA and p38 signaling stabilizes the MT network.238 Alternatively, signaling may enhance dynein– dynactin binding to hexon and boost the fast microtubule-dependent motions.225,234,239

6.2  Deoxyribonucleic Acid Uncoating and Nuclear Import How viral capsids transit from MT to the NPC is poorly understood. Yet, imaging by fluorescence and electron microscopy has shown that the viral DNA is in the context of a hexon-containing coat when it reaches the NPC. Accordingly, small compounds or antihexon antibodies blocked the translocation of cytosolic virus to the NPC.240–242 A key factor for the association of the incoming virus with the NPC is the nuclear export factor chromosome region of maintenance 1 (CRM1). Inhibition of CRM1 by leptomycin B, ratjandone A, or RNA interference strikingly arrested incoming viruses

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at the MTOC.240 It is possible that CRM1-mediated nuclear export is required for HAdV-C2/5 to dock to the NPC, that it facilitates a special type of transport from the MTOC to the NPC, or that it discharges viruses from MT proximal to the NPC. Docking of the partly uncoated HAdV capsids to the NPC depends on the nucleoporin Nup214.243,244 Depletion of Nup214 blocked HAdV-C5 nuclear targeting, whereas expression of the N-terminal 137 amino acids of Nup214 restored virus binding. The maximal transport diameter of the NPC is about 40 nm, which excludes incoming AdV from the nucleus.245 To overcome this size restriction, the DNA is uncoated before being imported into the nucleus. For HAdV-C2/5, this occurs through the recruitment of conventional kinesin-1.246 When AdVs are docked to the NPC, both protein XI and Nup358 engage with kinesin-1 via the kinesin light and heavy chain. Kinesin motions provide pulling forces that disrupt capsids and release viral DNA. Consequently, the nucleoporins Nup358, Nup68, and Nup214 are displaced to the cytoplasm with parts of the uncoated capsids. Partly disassembled NPCs show increased permeation of fluorescent dextrans, and are probably permissive to viral DNA import. Further cellular factors are implicated in the import of viral DNA, including histone H1, heat-shock cognate protein 70, and importins α, β1, β2, and 7.243,247–249 Tracking of single viral DNA using copper(I)-catalyzed azide-alkyne cyclo-addition (click) chemistry and super-resolution microscopy revealed that, surprisingly, only 6–48% of viral genomes were successfully imported into the nucleus, and 25% of viral DNA was spread throughout the cytosol with large cell-to-cell variability.232 Future work employing single-genome tracking will further elaborate on mechanisms and implications of nuclear import and misdelivery of HAdV genomes (see also Ref. 250).

7.  Conclusions and Perspectives Adenoviruses have been known for more than 50 years. Yet, there are many open questions regarding how these viruses interact with their host during entry. We estimate that close to 100 host factors are involved in AdV entry. In addition, unresolved questions include the nature of virus, for example, the location of the minor cementing proteins, and the mode of genome encapsidation. The latter may affect the mechanisms of viral DNA unpacking during entry. Important questions regarding virus–host interactions include how the virus overcomes the hurdles toward long-lasting gene expression from adenovirus vectors. Answers to these questions are the basis for taking best advantage of the highly immunogenic nature of certain human AdVs and killing cancer cells. The aims involve strategies to de-target the virus from immune cells and the liver, to shield it from blood factors, target it to relevant cells, and finally arm it with genes of interest. Whereas re-targeting efforts are advanced, shielding is still in its infancy. It awaits the development of tunable coats that can respond to local cues of target cells: for example, by exposing the virus to the uncoating cues that determine efficient entry and genome delivery. Promising and versatile shielding and re-targeting strategies have exploited designed ankyrin repeats that can be selected for virtually any target at high affinity and selectivity.251,252 Alternatively, single-chain variable fragments, immunoglobulins

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from camelids, and derivatives from the latter have also been developed.253 Regarding immunity, key problems for the near future are to elucidate immunogenic features of AdVs and determine the mechanisms of how they occur in vitro and in vivo.254 A major challenge is to translate the findings from in vitro studies and animal studies to humans. It will be interesting to see whether clinical trials with oncolytic AdVs can close this gap or create new puzzles that require new technologies to be resolved.

Acknowledgments We thank Maarit Suomalainen and Justin Flatt for comments on the manuscript. The work was supported by the Swiss National Science Foundation (grant 31003A_141222/1 to UFG) and the Kanton Zurich.

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