Novel Fiber-Dependent Entry Mechanism for Adenovirus Serotype 5 in LacrimalAcini

ABSTRACT The established mechanism for infection of most cells with adenovirus serotype 5 (Ad5) involves fiber capsid protein binding to coxsackievirus-adenovirus receptor (CAR) at the cell surface, followed by penton base capsid protein binding to αv integrins, which triggers clathrin-mediated endocytosis of the virus. Here we determined the identity of the capsid proteins responsible for mediating Ad5 entry into the acinar epithelial cells of the lacrimal gland. Ad5 transduction of primary rabbit lacrimal acinar cells was inhibited by excess Ad5 fiber or knob (terminal region of the fiber) but not excess penton base. Investigation of the interactions of recombinant Ad5 penton base, fiber, and knob with lacrimal acini revealed that the penton base capsid protein remained surface associated, while the knob domain of the fiber capsid protein was rapidly internalized. Introduction of rabbit CAR-specific small interfering RNA (siRNA) into lacrimal acini under conditions that reduced intracellular CAR mRNA significantly inhibited Ad5 transduction, in contrast to a control (nonspecific) siRNA. Preincubation of Ad5 with excess heparin or pretreatment of acini with a heparinase cocktail each inhibited Ad5 transduction by a separate and apparently additive mechanism. Functional and imaging studies revealed that Ad5, fiber, and knob, but not penton base, stimulated macropinocytosis in acini and that inhibition of macropinocytosis significantly reduced Ad5 transduction of acini. However, inhibition of macropinocytosis did not reduce Ad5 uptake. We propose that internalization of Ad5 into lacrimal acini is through a novel fiber-dependent mechanism that includes CAR and heparan sulfate glycosaminoglycans and that the subsequent intracellular trafficking of Ad5 is enhanced by fiber-induced macropinocytosis.

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Integrinα3β1 Is an Alternative Cellular Receptor for AdenovirusSerotype5

Journal of Virology ◽

10.1128/jvi.77.24.13448-13454.2003 ◽

2003 ◽

Vol 77 (24) ◽

pp. 13448-13454 ◽

Cited By ~ 44

Author(s):

Barbara Salone ◽

Yuri Martina ◽

Stefania Piersanti ◽

Enrico Cundari ◽

Gioia Cherubini ◽

...

Keyword(s):

Binding Motif ◽

Penton Base ◽

Cell Binding ◽

Binding Interface ◽

Serotype 5 ◽

Blocking Antibodies ◽

Adenovirus Receptor ◽

Integrin Α3β1

ABSTRACT Many adenovirus serotypes enter cells by high-affinity binding to the coxsackievirus-adenovirus receptor (CAR) and integrin-mediated internalization. In the present study, we analyzed the possible receptor function of α3β1 for adenovirus serotype 5 (Ad5). We found that penton base and integrin α3β1 could interact in vitro. In vivo, both Ad5-cell binding and virus-mediated transduction were inhibited in the presence of anti-α3 and anti-β1 function-blocking antibodies, and this occurred in both CAR-positive and CAR-negative cell lines. Peptide library screenings and data from binding experiments with wild-type and mutant penton base proteins suggest that the Arg-Gly-Asp (RGD) in the penton base protein, the best known integrin binding motif, is only part of the binding interface with α3β1, which involved multiple additional contact sites.

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Adenovirus Serotype 30 Fiber Does Not Mediate Transduction via the Coxsackie-Adenovirus Receptor

Journal of Virology ◽

10.1128/jvi.76.2.656-661.2002 ◽

2002 ◽

Vol 76 (2) ◽

pp. 656-661 ◽

Cited By ~ 11

Author(s):

Lane K. Law ◽

Beverly L. Davidson

Keyword(s):

Viral Entry ◽

Cho Cells ◽

Chinese Hamster ◽

Wild Type Virus ◽

Adenovirus Serotype ◽

Coxsackie Adenovirus Receptor ◽

Serotype 5 ◽

Overlap Pcr ◽

Adenovirus Receptor ◽

Group D

ABSTRACT Prior work by members of our laboratory and others demonstrated that adenovirus serotype 30 (Ad30), a group D adenovirus, exhibited novel transduction characteristics compared to those of serotype 5 (Ad5, belonging to group C). While some serotype D adenoviruses bind to the coxsackie-adenovirus receptor (CAR), the ability of Ad30 fiber to bind CAR is unknown. We amplified and purified Ad30 and cloned the Ad30 fiber by overlap PCR. Alignment of Ad30 fiber with Ad3, Ad35, Ad5, Ad9, and Ad17 revealed that Ad30, like Ad9 and Ad17, has a shortened fiber sequence relative to that of Ad5. The knob region of fiber was 45% identical to that of the Ad5 knob regions. We made a chimeric recombinant virus (Ad5GFPf30) in which the Ad5 fiber (amino acids [aa]47 to 582) was replaced with Ad30 fiber sequences (aa 46 to 372), and CAR-mediated viral entry was determined on CAR-expressing Chinese hamster ovary (CHO) cells. While CAR expression significantly increased Ad5GFP-mediated transduction in CHO cells (from 1 to 36%), it did not enhance Ad5GFPf30 gene transfer. Binding of radiolabeled Ad5GFPf30 or Ad30 wild-type virus was also not improved by the expression of CAR. These results suggest that Ad30 fiber is distinct from Ad5, Ad9, and Ad17 fibers in its inability to direct transduction via CAR.

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Combined Fiber Modifications Both to Target αvβ6and Detarget the Coxsackievirus–Adenovirus Receptor Improve Virus Toxicity ProfilesIn Vivobut Fail to Improve Antitumoral Efficacy Relative to Adenovirus Serotype 5

Human Gene Therapy ◽

10.1089/hum.2011.218 ◽

2012 ◽

Vol 23 (9) ◽

pp. 960-979 ◽

Cited By ~ 25

Author(s):

Lynda Coughlan ◽

Sabari Vallath ◽

Alena Gros ◽

Marta Giménez-Alejandre ◽

N. Van Rooijen ◽

...

Keyword(s):

Adenovirus Serotype ◽

Serotype 5 ◽

Adenovirus Receptor

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Adenovirus serotype 5 infects human dendritic cells via a coxsackievirus–adenovirus receptor-independent receptor pathway mediated by lactoferrin and DC-SIGN

Journal of General Virology ◽

10.1099/vir.0.008342-0 ◽

2009 ◽

Vol 90 (7) ◽

pp. 1600-1610 ◽

Cited By ~ 40

Author(s):

William C. Adams ◽

Emily Bond ◽

Menzo J. E. Havenga ◽

Lennart Holterman ◽

Jaap Goudsmit ◽

...

Keyword(s):

Dendritic Cells ◽

Viral Entry ◽

Cell Activation ◽

Viral Vector ◽

Antibacterial Properties ◽

Minor Role ◽

Adenovirus Serotype ◽

Serotype 5 ◽

Plasmacytoid Dcs ◽

Adenovirus Receptor

The coxsackievirus–adenovirus receptor (CAR) is the described primary receptor for adenovirus serotype 5 (Ad5), a common human pathogen that has been exploited as a viral vector for gene therapy and vaccination. This study showed that monocytes and dendritic cells (DCs), such as freshly isolated human blood myeloid DCs, plasmacytoid DCs and monocyte-derived DCs, are susceptible to recombinant Ad5 (rAd5) infection despite their lack of CAR expression. Langerhans cells and dermal DCs from skin expressed CAR, but blocking CAR only partly decreased rAd5 infection, together suggesting that other receptor pathways mediate viral entry of these cells. Lactoferrin (Lf), an abundant protein in many bodily fluids known for its antiviral and antibacterial properties, promoted rAd5 infection in all cell populations except plasmacytoid DCs using a CAR-independent process. Lf caused phenotypic differentiation of the DCs, but cell activation played only a minor role in the increase in infection frequencies. The C-type lectin receptor DC-SIGN facilitated viral entry of rAd5–Lf complexes and this was dependent on high-mannose-type N-linked glycans on Lf. These results suggest that Lf present at high levels at mucosal sites can facilitate rAd5 attachment and enhance infection of DCs. A better understanding of the tropism and receptor mechanisms of Ad5 may help explain Ad5 pathogenesis and guide the engineering of improved rAd vectors.

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Conformational Changes in the Adenovirus Hexon Subunit Responsible for Regulating Cytoplasmic Dynein Recruitment

Journal of Virology ◽

10.1128/jvi.02889-14 ◽

2014 ◽

Vol 89 (2) ◽

pp. 1013-1023 ◽

Cited By ~ 15

Author(s):

Julian Scherer ◽

Richard B. Vallee

Keyword(s):

Capsid Protein ◽

Conformational Changes ◽

Structural Changes ◽

Motor Protein ◽

Cytoplasmic Dynein ◽

Low Ph ◽

Virus Infectivity ◽

Adenovirus Serotype ◽

Serotype 5 ◽

Ph Dependent

ABSTRACTVirus capsids provide genome protection from environmental challenges but are also poised to execute a program of compositional and conformational changes to facilitate virion entry and infection. The most abundant adenovirus serotype 5 (AdV5) capsid protein, hexon, directly recruits the motor protein cytoplasmic dynein following virion entry. Dynein recruitment is crucial for capsid transport to the nucleus and requires the transient exposure of AdV5 hexon to low pH, presumably mimicking passage through the endosomal compartment. These results suggest a pH-dependent capsid modification during early infection. The changes to hexon structure controlling this behavior have not been explored. We report that hexon remains trimeric at low pH but undergoes more subtle conformational changes. These changes are indicated by increased sensitivities to SDS-mediated dissociation and dispase proteolysis. Both effects are reversed at neutral pH, as is dynein binding by low-pH-treated hexon. Dispase cleavage, which we find maps to a specific site within hypervariable region 1 (HVR1) of AdV5 hexon, has no apparent effect on virion entry but completely inhibits its transport to the nucleus. In addition, an AdV5 mutant containing HVR1 of AdV48 is unable to bind dynein and is strongly inhibited in the postentry transport step. These results reveal that conformational changes involving hexon HVR1 are the basis for a novel viral mechanism controlling capsid transport to the nucleus.IMPORTANCEThe adenovirus serotype 5 (AdV5) capsid protein hexon recruits the molecular motor protein cytoplasmic dynein in a pH-dependent manner, a function critical for efficient transport toward the nucleus and AdV5 infectivity. In this work, we describe how low-pH exposure induces reversible structural changes in AdV5 hexon and how these changes affect dynein binding. In addition, we identified a pH-sensitive dispase cleavage site in hexon HVR1, which depends on the same structural changes and furthermore regulates dynein recruitment and capsid redistribution in infected cells. These data provide the first evidence relating long-known but subtle pH-dependent structural changes in hexon to a more recently established essential but poorly understood role in virus transport. These results have broad implications for understanding virus infectivity in general, and our ability to block the recruitment mechanism has potential therapeutic implications as well.

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