Cell Entry and Trafficking of Human Adenovirus Bound to Blood Factor X Is Determined by the Fiber Serotype and Not Hexon:Heparan Sulfate Interaction

ABSTRACTOne of the first lines of host defense against many viruses in vertebrates is the innate immune system, which detects pathogen-associated molecular patterns (PAMPs) using pathogen recognition receptors (PRR). The dynamic interactions between pathogens and hosts create, in some cases, species-specific relationships. Recently, it was shown that murine factor X (mFX)-armored human adenovirus (HAd) stimulated a mFX-Toll-like receptor 4 (TLR4)-associated response in mouse macrophagesin vitroandin vivo. Given the importance of studies using animals to better understand host-pathogen interactions, we asked if human FX (hFX)-armored HAd type 5 (HAd5) was capable of activating innate immune sensors in primary human mononuclear phagocytes. To this end, we assayed human mononuclear phagocytes for their ability to be stimulated by hFX-armored HAd5 via a TLR/NF-κB pathway, in particular, a TLR4 pathway. In our hands, we found no significant interaction, activation, or maturation of human mononuclear phagocytes caused by the presence of hFX-armored HAd5.IMPORTANCEAnimals, and mice in particular, are often used as informative and powerful surrogates for how pathogens interact with natural host systems. When possible, extended and targeted studies in the natural host can then be performed. Our data will help us understand the differences in preclinical testing in mice and clinical use in humans in order to improve treatment for HAd diseases and Ad vector effectiveness.

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The Fiber Knob Protein of Human Adenovirus Type 49 Mediates Highly Efficient and Promiscuous Infection of Cancer Cell Lines Using a Novel Cell Entry Mechanism

Journal of Virology ◽

10.1128/jvi.01849-20 ◽

2020 ◽

Author(s):

Alexander T. Baker ◽

James A. Davies ◽

Emily A. Bates ◽

Elise Moses ◽

Rosie M. Mundy ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Surface Potential ◽

Human Adenovirus ◽

Cancer Cell Lines ◽

Cell Entry ◽

Therapeutic Applications ◽

Wide Range ◽

Pseudotyped Vector ◽

Entry Mechanism

The human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied. Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, while potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumor types. IMPORTANCE Adenoviruses are powerful tools experimentally and clinically. To maximize efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focused on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism. We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

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UNDERESTIMATED INFECTION - ON THE QUESTION OF THE HUMAN ADENOVIRUS PATHOGENICITY FACTORS

Voprosy Virusologii ◽

10.18821/0507-4088-2019-64-2-53-62 ◽

2019 ◽

Vol 64 (2) ◽

pp. 53-62

Author(s):

M. R. Ageeva ◽

S. B. Yatsyshina

Keyword(s):

Respiratory Diseases ◽

Coagulation Factor ◽

Human Adenovirus ◽

Adenovirus Infection ◽

Factor X ◽

Blood Proteins ◽

Lethal Outcome ◽

Cellular Receptors ◽

Pathogenicity Factors ◽

Severity Of The Disease

Human adenoviruses cause different organ infections of varying severity, from asymptomatic to severe cases with lethal outcome, that are registered everywhere. Detailed and focused study of factors predisposing to a severe course of infection is required. The literature contains information indicating the association of severe adenoviral respiratory diseases with certain types of adenovirus, primarily type 7. This review highlights the possible causes of increased pathogenicity of some types of adenovirus and their association with severe forms of infection. Pathogenicity factors include the ability of adenovirus to bind the specific cellular receptors, the formation of subviral particles, the interaction with blood proteins, in particular the coagulation factor X, as well as the features of the early genes E1A, E1B, E3, E4. In addition, the severity of the disease may be affected by the presence or absence of pre-existing antibodies specific to certain types of adenoviruses. Chronic diseases or immunosuppression also increase the risk of severe adenovirus infection. The information presented in this review may elucidate the pathogenesis of adenovirus infection, and help to develop new features for prevention and treatment.

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Cryo-Em Imaging of a Pseudotyped Adenovhtus with Ocular Cell Tropism

Microscopy and Microanalysis ◽

10.1017/s1431927600033924 ◽

2000 ◽

Vol 6 (S2) ◽

pp. 286-287

Author(s):

P.L. Stewart ◽

C.Y. Chiu ◽

D. Von Seggern ◽

G.R. Nemerow

Keyword(s):

Viral Entry ◽

Human Adenovirus ◽

Cell Entry ◽

Soluble Form ◽

Cell Tropism ◽

Penton Base ◽

Single Particle Reconstruction ◽

Virus Particles ◽

Entry Pathway ◽

Reconstruction Methods

Human adenovirus (Ad) provides unique model system for studying the multistage events in viral entry. We are applying cryo-electron microscopy and single particle reconstruction methods to study the cell entry pathway of Ad as well as to examine the structure of reengineered Ad vectors. Ad cell entry is mediated by interactions with two different cellular receptors: the fiber receptor, CAR for most Ad serotypes or a 50 kDa receptor for a subset of serotypes including Ad37, for attachment; and the αvβ3 and αvβ5 integrins for internalization. We have previously reconstructed both Ad2 and Adl2 virus particles complexed with a soluble form of αvβ5 integrin. We learned during our comparative cryo-EM study of the Ad2/αvβ5 and Adl2/αvβ5 complexes that the length of the variable RGD loop in the penton base affects the relative height of the integrin density over the penton base.

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The fiber knob protein of human adenovirus type 49 mediates highly efficient and promiscuous infection of cancer cell lines using a novel cell entry mechanism

10.1101/2020.07.20.213223 ◽

2020 ◽

Author(s):

Alexander T. Baker ◽

Gareth Marlow ◽

James A. Davies ◽

Elise Moses ◽

Rosie M. Mundy ◽

...

Keyword(s):

Cell Lines ◽

Cancer Cell ◽

Surface Potential ◽

Human Adenovirus ◽

Cancer Cell Lines ◽

Cell Entry ◽

Therapeutic Applications ◽

Wide Range ◽

Pseudotyped Vector ◽

Entry Mechanism

AbstractThe human adenovirus (HAdV) phylogenetic tree is diverse, divided across seven species and comprising over 100 individual types. Species D HAdV are rarely isolated with low rates of pre-existing immunity, making them appealing for therapeutic applications. Several species D vectors have been developed as vaccines against infectious diseases where they induce robust immunity in pre-clinical models and early phase clinical trials. However, many aspects of the basic virology of species D HAdV, including their basic receptor usage and means of cell entry, remain understudied.Here, we investigated HAdV-D49, which previously has been studied for vaccine and vascular gene transfer applications. We generated a pseudotyped HAdV-C5 presenting the HAdV-D49 fiber knob protein (HAdV-C5/D49K). This pseudotyped vector was efficient at infecting cells devoid of all known HAdV receptors, indicating HAdV-D49 uses an unidentified cellular receptor. Conversely, a pseudotyped vector presenting the fiber knob protein of the closely related HAdV-D30 (HAdV-C5/D30K), differing in four amino acids to HAdV-D49, failed to demonstrate the same tropism. These four amino acid changes resulted in a change in isoelectric point of the knob protein, with HAdV-D49K possessing a basic apical region compared to a more acidic region in HAdV-D30K. Structurally and biologically we demonstrate that HAdV-D49 knob protein is unable to engage CD46, whilst potential interaction with CAR is extremely limited by extension of the DG loop. HAdV-C5/49K efficiently transduced cancer cell lines of pancreatic, breast, lung, oesophageal and ovarian origin, indicating it may have potential for oncolytic virotherapy applications, especially for difficult to transduce tumour types.ImportanceAdenoviruses are powerful tools experimentally and clinically. To maximise efficacy, the development of serotypes with low pre-existing levels of immunity in the population is desirable. Consequently, attention has focussed on those derived from species D, which have proven robust vaccine platforms. This widespread usage is despite limited knowledge in their basic biology and cellular tropism.We investigated the tropism of HAdV-D49, demonstrating it uses a novel cell entry mechanism that bypasses all known HAdV receptors. We demonstrate, biologically, that a pseudotyped HAdV-C5/D49K vector efficiently transduces a wide range of cell lines, including those presenting no known adenovirus receptor. Structural investigation suggests that this broad tropism is the result of a highly basic electrostatic surface potential, since a homologous pseudotyped vector with a more acidic surface potential, HAdV-C5/D30K, does not display a similar pan-tropism. Therefore, HAdV-C5/D49K may form a powerful vector for therapeutic applications capable of infecting difficult to transduce cells.

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Human adenovirus type 17 from species D transduces endothelial cells and human CD46 is involved in cell entry

Scientific Reports ◽

10.1038/s41598-018-31713-x ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 5

Author(s):

Jing Liu ◽

Philip Boehme ◽

Wenli Zhang ◽

Jun Fu ◽

Roma Yumul ◽

...

Keyword(s):

Endothelial Cells ◽

Human Adenovirus ◽

Adenovirus Type ◽

Cell Entry

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Development of a Targeted Gene Vector Platform Based on Simian Adenovirus Serotype 24

Journal of Virology ◽

10.1128/jvi.02425-09 ◽

2010 ◽

Vol 84 (19) ◽

pp. 10087-10101 ◽

Cited By ~ 10

Author(s):

Natalya Belousova ◽

Galina Mikheeva ◽

Chiyi Xiong ◽

Suren Soghomonian ◽

Daniel Young ◽

...

Keyword(s):

Gene Delivery ◽

Human Tumor ◽

Coagulation Factor ◽

Human Adenovirus ◽

High Rate ◽

Specific Gene ◽

Factor X ◽

Adenovirus Serotype ◽

Genetic Interventions

ABSTRACT Efforts to develop adenovirus vectors suitable for genetic interventions in humans have identified three major limitations of the most frequently used vector prototype, human adenovirus serotype 5 (Ad5). These limitations—widespread preexisting anti-Ad5 immunity in humans, the high rate of transduction of normal nontarget tissues, and the lack of target-specific gene delivery—justify the exploration of other Ad serotypes as vector prototypes. In this paper, we describe the development of an alternative vector platform using simian Ad serotype 24 (sAd24). We found that sAd24 virions formed unstable complexes with blood coagulation factor X and, because of that, transduced the liver and other organs at low levels when administered intravenously. The overall pattern of biodistribution of sAd24 particles was similar, however, to that of Ad5, and the intravenously injected sAd24 was cleared by Kupffer cells, leading to their depletion. We modified the virus's fiber protein to design a Her2-specific derivative of sAd24 capable of infecting target human tumor cells in vitro. In the presence of neutralizing anti-Ad5 antibodies, Her2-mediated infection with targeted sAd24 compared favorably to that with the Ad5-derived vector. When used to target Her2-expressing tumors in animals, this fiber-modified vector achieved a higher level of gene transfer to metastasis-containing murine lungs than to tumor-free lungs. In aggregate, these studies provide important insights into sAd24 biology, identify its advantages and limitations as a vector prototype, and are thus essential for further development of an sAd24-based gene delivery platform.

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Identification of novel human adenovirus candidates using the coxsackievirus and adenovirus receptor for cell entry

Virology Journal ◽

10.1186/s12985-020-01318-w ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Kemal Mese ◽

Oskar Bunz ◽

Sebastian Schellhorn ◽

Wolfram Volkwein ◽

Dominik Jung ◽

...

Keyword(s):

Human Adenovirus ◽

Cell Entry ◽

Coxsackievirus And Adenovirus Receptor ◽

Adenovirus Receptor

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Direct Evidence from Single-Cell Analysis that Human α-Defensins Block Adenovirus Uncoating To Neutralize Infection

Journal of Virology ◽

10.1128/jvi.02471-09 ◽

2010 ◽

Vol 84 (8) ◽

pp. 4041-4049 ◽

Cited By ~ 62

Author(s):

Emily K. Nguyen ◽

Glen R. Nemerow ◽

Jason G. Smith

Keyword(s):

Direct Evidence ◽

Single Cell Analysis ◽

Single Cells ◽

Indirect Evidence ◽

Human Adenovirus ◽

Cell Entry ◽

Adenovirus Infection ◽

Viral Genomes ◽

Virus Particles ◽

Evolutionarily Conserved

ABSTRACT Human α-defensins are evolutionarily conserved effectors of the innate immune response with broadly acting antibacterial activity. Their role in antiviral immunity is less well understood. We previously showed that these antimicrobial peptides are potent inhibitors of human adenovirus infection. Based on biochemical studies and indirect evidence from confocal microscopy, we proposed that defensins bind to and stabilize the virus capsid and neutralize infection by preventing the release of the endosomalytic protein VI. To determine whether defensin action also restricts exposure of the viral genome, we developed a system to evaluate adenovirus uncoating during cell entry by monitoring the exposure of BrdU-labeled viral genomes. This assay allowed us to determine the kinetics of uncoating of virus particles in single cells. Using this assay, we now provide direct evidence that human α-defensins block adenovirus infection by preventing uncoating during cell entry.

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