| 1. |
- Chandra, Naresh, 1987-
(författare)
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The glycobiology of human adenovirus infections : implications for tropism and treatment
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human adenoviruses (HAdVs) are common human pathogens, causing gastrointestinal, ocular, and respiratory infections on a regular basis. Epidemic keratoconjunctivitis (EKC) is a severe ocular infection for which no approved antivirals are available. HAdV-D37 is one of the causative agents of EKC and uses sialic acid (SA)-containing glycans as cellular receptors. HAdV-D37 interacts with SA via the knob domain of the trimeric virus fiber protein, containing three SA-binding sites. HAdV-D37 also bind to glycosaminoglycans (GAGs), but the outcome of this interaction remains unknown. Here, using biochemical and cell-based assays, the impact of GAGs on HAdV-D37 infection (paper I) was investigated. We found that HAdV-D37 interacts with both soluble and cell-surface sulfated GAGs via the knob domain of the viral fiber protein. Remarkably, removal of heparan sulfate (HS; a type of GAG) from human corneal epithelial (HCE) cells by heparinase III enhanced HAdV-D37 infection. We propose that sulfated GAGs in bodily secretions and on plasma membranes function as decoy receptors that prevent the virus from binding to SA-containing receptors and inhibit subsequent virus infection. We also found abundant HS in the basement membrane of the human corneal epithelium. We suggest that this layer of HS functions as a barrier to sub-epithelial infection of HAdV-D37. Based on this finding, we hypothesized that GAG-mimetics may act as artificial decoy receptors and inhibit HAdV-D37 infection. Here, the antiviral effect of suramin (a known GAG-mimetic) and its analogs against HAdV-D37 (paper II) was evaluated. Interestingly, all compounds displayed antiviral effects by inhibiting the binding of HAdV-D37 to HCE cells. The antiviral effect of suramin was HAdV species-specific. We report for the first time that virus binding to cell-surface decoy receptor constitutes a potential target for antiviral drug development.HAdVs are the major cause of infectious conjunctivitis, constituting up to 75% of all conjunctivitis cases worldwide. Species B HAdV type 3 (HAdV-B3) causes pharyngoconjunctival fever (PCF), whereas HAdV-D8, -D37, and -D64 cause EKC. Recently, HAdV-D53, -D54, and -D56 have emerged as new EKC-causing agents. HAdV-E4 causes both PCF and EKC. SA-containing glycans have been established as cellular receptors for HAdV-D37. By means of cell-based assays, we investigated if ocular HAdVs other than HAdV-D37 also use SA-containing glycans as receptors on HCE cells (paper III). It was found that SA-containing glycans function as cellular receptors for five (HAdV-D8, -D37, -D53, -D54, and -D64) out of six EKC-causing species D HAdVs. We showed that these viruses interact with SAs via the knob domain of the viral fiber protein. HAdV-E4 and -D56 infection of cells was independent of SAs. Surprisingly, HCE cells were completely refractory to HAdV-B3 infection. A trivalent sialic acid (TSA) derivative ME0462 (compound 17a in paper II), designed to bind to SA-binding sites on HAdV-D37 fiber knob, also showed potent antiviral activity against several EKC-causing HAdVs. This suggests that ME0462 can be used as a broad-spectrum antiviral against known and emerging EKC-causing HAdVs. Surface plasmon resonance (SPR) analysis confirmed a direct interaction between ME0462 and fiber knobs of EKC-causing HAdVs.Recently, a TSA derivative (ME0322; designed to bind to SA-binding sites on HAdV-D37 fiber knob) was shown potent antiviral against HAdV-D37 in vitro. To improve the antiviral potency of this compound, six new TSA derivatives were synthesized and their inhibitory effects were evaluated against HAdV-D37 (paper IV). Interestingly, the best compound 17a was found approximately three orders of magnitude more potent (IC50 (binding) = 1.4 nM, IC50 (infection) = 2.9 nM) than ME0322 (IC50 in µM range). SPR data showed that HAdV-D37 fiber knob binds to TSA compounds with high affinities. Structural data revealed the trivalent binding mode of all newly synthesized TSA compounds to HAdV-D37 fiber knob. Ophthalmic toxicity of compound 17a (best compound) was also investigated in rabbits without any sign of toxicity.HAdV-D36 is a member of species D HAdV and has the ability to infect a broad range of animals, which is unusual for HAdVs. Another remarkable feature of HAdV-D36 is that this virus induces obesity in experimental animals. Several epidemiological studies highlighted a link between HAdV-D36 and human obesity. There is no information about the cellular receptor usage by HAdV-D36. Using structural biology and cell-based approaches, we investigated the cellular receptor(s) for HAdV-D36 (paper V). We show that HAdV-D36 attaches to host cells (via the fiber knob) using the coxsackie and adenovirus receptor (CAR), SA-containing glycans, and one or more unknown proteins or glycoproteins. Using glycan microarray, we found that HAdV-D36 displays binding preference to a rare SA-variant: 4-O,5-N-diacetylneuraminic acid (Neu4,5Ac2), over the more common SA (in humans) i.e. 5-N-acetylneuraminic acid (Neu5Ac). Structural analysis of HAdV-D36 fiber knob:Neu4,5Ac2 complex explained this preference. To date, Neu4,5Ac2 has not been detected in humans, although it is synthesized by many domestic and livestock animals. Our results indicate that HAdV-D36 has evolved to utilize a specialized set of cellular receptors that coincide with a unique host range and pathogenicity profile.These studies provide insights into multiple roles of glycans in HAdV infection cycle and highlight the therapeutic potential of glycans/glycan-mimetics in HAdV-D37 infection.
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| 2. |
- Rajan, Anandi, 1988-
(författare)
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Capsid protein functions of enteric human adenoviruses
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Human adenoviruses (HAdVs) cause respiratory illnesses, epidemic conjunctivitis and infantile gastroenteritis. HAdV types 40 and 41 cause enteric infections in infants worldwide. HAdVs use various receptors for attachment onto different host cells. Coxsackievirus and adenovirus receptor, CD46, sialic acid, coagulation factors IX and X, lactoferrin and heparan sulfate are some receptors and molecules which the hexon and fiber proteins (components of the capsid) bind for direct or indirect cellular attachment. The penton base protein (another component of the capsid) is responsible for the internalization of the virus into the host cell. An arginine-glycine-aspartic acid amino acid motif is present in most but not all adenovirus penton base proteins and mediates interaction with αv integrins, resulting in internalization.The enteric HAdVs are unique since they do not have this arginine-glycine-aspartic acid motif on their penton base. Using a library of hamster cells expressing specific human integrins, along with recombinant soluble penton base from HAdV type 41 and commercially available soluble laminins, we identified laminin-binding integrins as co-receptors for entry and infection of human intestinal HT-29 cells by the enteric HAdVs.HAdV types 40, 41 and 52 are the only three HAdVs that have two different fiber proteins, one long and one short. By performing cell binding and infection experiments, we have found that the receptor for the short fiber of HAdV-52 is sialic acid-containing glycans and the long fiber receptor is CAR although most of the binding was dependent on sialic acid-containing glycans. We also observed that the short fiber of HAdV type 40 interacts with soluble heparin or cell surface heparan sulfate. Further investigation pointed out that the specific sulfate groups on heparin/heparan sulfate (sulfated glycosaminoglycans) are important for this binding. Also, we identified that the interaction and utilization of these glycosaminoglycans as receptors is dependent on exposure to low pH. We also studied the potential mechanism behind the symptoms caused by these enteric HAdVs in enteroendocrine cells called enterochromaffin cells. We could show that the short fiber and the hexon of HAdV type 41 stimulated release of serotonin from the enterochromaffin cells, which can be a cause of vomiting and diarrhea.These studies have given us insight into the role of enteric HAdV capsid proteins as ligands to hitherto unidentified receptors and co-receptors. We also show that these molecules play important functions in the virus’ infectious cycle and probably also in their disease mechanism of host cells.
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| 3. |
- Storm, Rickard, 1986-
(författare)
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Early host cell interactions and antivirals against ocular adenoviruses
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Viruses are common causative agents of ocular infection among humans. Epidemic keratoconjuntivitis (EKC) is a severe and contagious ocular disease with reported outbreaks worldwide. It is estimated that this disease affects 20-40 million individuals every year, which leads to huge socioeconomic costs for the affected countries. EKC is characterized by keratitis and conjunctivitis but is also associated with pain, edema, lacrimation, and decreased vision that can prolong for months after the infection and in rare cases years. This disease is caused by human adenoviruses (HAdVs), which belong to the family of Adenoviridae. Currently, there is no available treatment against EKC.EKC is mainly caused by HAdV-8, HAdV-19, HAdV-37, HAdV-53, HAdV-54, and HAdV-56, which belong to species D HAdVs. HAdV-8, HAdV-19 and HAdV-37 have previously been shown to use sialic acid (SA)-containing glycans as cellular receptors to bind to and infect human corneal epithelial (HCE) cells. To characterize the receptor in more detail, we performed a glycan array, which included SA-containing glycans. A branched hexasaccharide terminating with SA in each arm was identified as a candidate receptor. This glycan corresponds to the glycan motif found on a ganglioside, GD1a. By performing a series of biological and biochemical experiments we confirmed the function of the GD1a glycan as a cellular receptor for EKC-causing HAdVs. However, the glycan used as a receptor was linked to plasma membrane protein(s) through O-glycosidic bonds, rather than to a lipid (as in the ganglioside). X-ray crystallography analysis showed that the two terminal SA:s interacted with two of the three previously identified SA-binding sites on the knob domain of the HAdV-37 capsid protein known as the fiber.Based on the structural features of the GD1a:HAdV-37 knob interaction, we assumed that a three-armed molecule with each arm terminating with SA would be an efficient inhibitor. Such molecules were designed, synthesized and found to efficiently prevent HAdV-37 binding to and infection of corneal cells. These results indicate that trisialic acids-containing compounds may be used for treatment of EKC.After binding to its primary receptor, most HAdVs have been shown to interact with αVβ3 and αVβ5 integrins to enter human cells. This interaction occurs through the RGD (arginine-alanine-aspartic acid) motif in the capsid protein known as the penton base. However, it was not clear if corneal epithelial cells express αVβ3 and αVβ5 integrins. Thus, to better understand additional early steps of infection by EKC-causing HAdVs, we performed binding and infection competition experiments using human corneal epithelial cells and siRNA, integrin specific antibodies, peptides and RGD-containing ligands indicating that α3, αV, β1 affected HAdV-37 infection of but not binding to HCE cells. We could also see that HAdV-37 co-localize with α3 and αV at after entry into HCE cells. In situ histochemistry confirmed that the expression of α3 and αV in human corneal tissue. Overall, our results suggest that αV and α3 integrins are important for HAdV-37 infection of corneal cells.Altogether, these results provide further insight into the biology of HAdVs and open up for development of novel antiviral drugs.
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| 4. |
- Upadhyay, Arunkumar, 1984-
(författare)
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Viperin, a multifunctional radical SAM enzyme : biogenesis and antiviral mechanisms
- 2016
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Viperin (virus-inhibitory protein, endoplasmic reticulum-associated, interferon-inducible) is an interferon-induced antiviral protein. It has three distinct functional domains: the N-terminal domain, the radical SAM (S-adenosylmethionine) domain for binding iron-sulphur cluster, and the C-terminus domain. Viperin has a broad antiviral effect, and is also involved in the immune response signalling. However, the function and antiviral mechanism of viperin are still not well characterized. Thus the overall aim of the thesis was to investigate and better understand the function of viperin and its antiviral mechanism by identifying the cellular network of interaction partners. Affinity purification and mass spectrometry analysis were used to identify cellular proteins that interact with viperin.CIA1 (also known as CIAO1), a factor involved in loading of iron-sulphur (Fe/S) cluster was identified and confirmed to interact at the C-terminus of viperin. It was also seen that the C-terminal domain and the functional SAM domain of viperin was essential for loading the Fe/S cluster onto viperin. On a closer look at the biogenesis of viperin, we identified and confirmed viperin interaction with CIA2A, CIA2B, (also known as FAM96A and FAM96B respectively) and MMS19, which are other factors involved in the transfer of Fe/S clusters onto cytosolic Fe/S apo-proteins. Surprisingly, MMS19, which has been shown to act as an adapter protein for other Fe/S proteins, only interacted indirectly and was not required for transferring the Fe/S cluster. Similarly, the interaction of viperin with both the isoforms of CIA2 was interesting, but the role they play in viperin biogenesis and antiviral function is still not clear and requires further investigation.Study of the antiviral action of viperin against tick-borne encephalitis virus (TBEV) showed that the activity of the SAM domain is essential for the strong inhibition of genome replication of TBEV. Furthermore, viperin also affects the assembly and release of TBEV. Viperin interacts with GBF1 and downregulates its activity, thus preferentially inducing the secretion of viral capsid protein from the cell, and therefore disrupting the formation of infectious virus particles. The N-terminal domain of viperin is important for its effect on assembly and release.In summary, this work contributes to our general understanding of viperin biogenesis in the cell regarding the loading of Fe/S cluster onto viperin. It also addresses the importance of the different domains for its antiviral function against TBEV. Finally, mass spectrometry and viperin interactome analysis implicate many other interesting cellular pathways or processes that might give us a better understanding of viperin’s function and antiviral mechanism.
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