As a result of transcriptional editing, Ebola virus (EBOV) produces multiple soluble products from its glycoprotein gene, the primary product of which is the secreted glycoprotein (sGP), in addition to the membrane-bound viral spike protein GP1,2. A lack of leukocyte infiltration is observed during EBOV infection, which is thought to allow virus replication to proceed unchecked and thus represents a significant role in the immunopathology of the disease. Currently the only know function of sGP is that it has an anti-inflammatory effect on endothelial cells treated with TNF-α, an effect that has been hypothesized to interfere with recruitment or extravasation of leukocytes. To better characterize this anti-inflammatory function, a link between sGP structure and function was sought. Mass spectrometry (MS) analysis of recombinant sGP demonstrated that it is a parallel-orientated disulphide-linked homodimer that contains Cys53-C53’ and Cys306-C306’ intermolecular disulphide bonds. In addition to being glycosylated with complex N-glycans, sGP also contained a novel post-translation modification, termed C-mannosylation. C-mannosylation was not required for the anti-inflammatory function of sGP; however, glycine mutations at amino acids 53 and 306 resulted in the complete loss of the anti-inflammatory effect on TNF-α treated endothelial cells. Thus, a specific structure mediated by intermolecular disulphide bonds is required for the proposed anti-inflammatory function of sGP, suggesting that this effect is the result of a specific interaction. The spike protein GP1,2, also contains C-mannosylation motifs. MS analysis of GP1,2 indicated that GP1 was C-mannosylated, while two adjacent motifs in the membrane proximal region (MPER) of GP2 were not. The infectious virus-like particle (iVLP) assay, a system for investigating virus particle assembly and entry, was utilized to determine the functional importance of these conserved tryptophans. Elimination of the C-mannosylation motif, which resides in an external loop region of GP1, increased reporter activity, suggesting that particle entry is enhanced and this region may interact with the cell surface despite being outside of the receptor binding site. Decreased reporter activity was observed for all MPER mutants, with multiple MPER tryptophan mutations resulting in decreased GP1,2 incorporation. These data place the MPER tryptophan residues in an important role for glycoprotein incorporation and particle entry. Given the tryptophan content and location is similar to the MPER of HIV gp41, where these residues are required for glycoprotein incorporation and fusion, the MPER of EBOV GP2 may function similarly.

The process of antigenic variation in African trypanosomes allows the survival of the parasite by constantly switching the variant surface glycoprotein (VSG) expressed in their surface. There are believed to be several hundred copies of these silent VSG genes in the parasite's genome and they are expressed differentially. The majority of these genes are not capable of being transcribed in situ and must therefore be expressed from specialised transcriptional units known as bloodstream expression sites (BESs). Only one such site is active at any one time, ensuring that a single VSG is expressed in the trypanosome's surface coat. Switching the expressed VSG involves replacing the VSG in the active BES, or activating a new BES in conjunction with silencing the previously active. Differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres. All BESs are telomeric and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with yeast telomeres that is directly involved in DNA recombination and gene silencing. The main aim of this thesis was to test the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. In order to compare trypanosome Ku homologues with those from other organisms, it was necessary to compile homology alignments with other Ku homologues using Clustal W analysis. Subsequent experiments looked at the fate of exogenously introduced restriction enzyme target sites after transient transformation with cassettes encoding the restriction enzyme. A final analysis looked for the presence of NHEJ in homologous recombination- deficient trypanosomes. Disrupting this element of DNA repair would hopefully lead to other forms of repair becoming detectable, and even up-regulated. Rad51, in yeast a member of the Rad52 epistasis group (integral in yeast homologous recombination), had previously been demonstrated to be involved in DNA repair in trypanosomes (McCulloch & Barry, 1999). rad51 mutants were electroporated with cassettes containing noncompatible ends that would prevent their integration into the endogenous genome via conventional homologous recombination. This cassette also contained promoter DNA sequence to allow selection in the event of integration into non-transcribed regions of the genome. Study of the junctions encompassing the integration sites of the cassette allowed investigation into how the cassettes were integrated, and revealed to us the extent of the sequence homology required to catalyse integration. The method of repair detection observed indicated that classical homologous recombination is not the only pathway utilised by African trypanosomes to metabolise DNA double-strand breaks.

Human cytomegalovirus (HCMV) is a ubiquitous pathogen with ability to establish a persistent infection in the host for many years. Diseases are more serious in congenitally infected infants and immunocompromised individuals such as AIDS patients and transplant recipients. It rarely causes disease among immunocompetent individuals. The envelope glycoproteins of HCMV (gB, gH, gN, gM, gL and gO) are essential for viral infectivity as they are involved in attachment and penetration of the host cell, cell to cell viral transmission and fusion of infected cells. Also, they are known to be important targets for humoral and cell mediated immune responses against the virus. The hypothesis states that HCMV genotypes are constantly mutating and evolving, then is controlled in a setting of immunocompetence, but in immunocompromised patients, congenitally infected patients or possibly within the CNS, the virus is able to evade the immune system and genotypes may evolve which are more pathogenic and/or neuroinvasive. In current study, a total of 669 CSF samples were collected from Malawian children with neurological illness that have been tested for the presence of HCMV. A comparison study population was also studied which contained 26 HCMV isolates obtained from the Manchester Royal Infirmary Clinical Virology Laboratory. The isolates were originally collected from congenitally CMV infected infants or immunocompromised children and adults showing symptoms of HCMV disease. Positive HCMV samples were screened for glycoproteins (B, H, N, L and O) and then glycotyped by restriction fragment length polymorphisms and sequencing analysis. HCMV DNA was found to be present in 46 (6.7%) out of 669 CSF samples. In 19 of these HCMV positive samples, glycoprotein genotypes (B, H, N, L and O) were identified. The predominant glycoprotein genotypes in the CSF population were as follows, gB type 3(63.2%), gH type 1(73.2%), gN type 4 (50%), gL type 4 (55.8%) and gO type 1 (60%), whilst in the comparison study population (control group) the most common glycoprotein genotypes were; gB type 3 (52.3%), gH type 1 (52%), gN type 4 (60%), gL type 4 (56%) and gO type 1(48%). No statistically significant difference was found between glycoprotein genotypes among the CSF samples and comparison study population. However, when linkage between glycoprotein types was studied and analysed differences between the two populations emerged. The particular glycoprotein linkage (gH1, gN1 and gO1) occured six times in the CSF study population with severe and fatal outcome as it occured four times with fatal outcome, and in two other patients, one was HIV positive and one with sequelae.Novel linkages between gO and gN genotypes were also found (gN3b/gO3), (gN4a/gO4), (gN4b/gO2b), (gN4b/gO3), (gN4d/gO1c), (gN1/gO1c), and (gN4c/gO4). The (gN1/gO1c) combination was occurred three times, but only in CSF study population and was associated with fatal outcomes, two patients died and one was HIV positive patients.RFLP assay was found to have limited efficacy in identification of HCMV genotypes, as discrepancies in genotype results between RFLP and sequencing were found in all glycoprotein genotypes. Thus, sequencing analysis was more accurate and recommended for identification of the HCMV genotypes.In conclusion, investigation of different glycoprotein genotypes in CSF samples has shown no significant correlation between single glycoprotein and disease outcome. Recombination between HCMV strains may lead to progression of disease. However, studying a large group of CSF samples positive for HCMV from immunocompromised patients may clarify the correlation between glycoprotein types and neurological outcome, and lead to improved strategies for treatment and prevention of HCMV associated disease.

Conclusions: CETA may be a more sensitive system than the bi-directional transport assay to detect transport of drugs with high passive diffusion across the BBB. We conclude that PHT, PB, OXC, ESL, CBZ-E, S-LC, and LCM, but not ESM, CBZ, RPM, ZNS, and PGB, are transported by human Pgp. These data suggest that resistance to PHT, PB, ESL, OXC and LCM might be attributed to increased efflux function of Pgp because they or their active metabolites are Pgp substrates. The CTC haplotype exhibited increased directional transport activity by Pgp. The effects of MDR1 polymorphisms on AED transport may provide a molecular explanation of the association between the polymorphisms and pharmacoresistance. This knowledge may help guide the design of genetic-based individualized therapy of epilepsy. / Methods: Stable transfected clones of human MDR1 haplotypes combining 1236C>T, 2677G>T/A, and 3435C>T in LLC-PK1 cells were established and validated. The expression level and localization of Pgp were measured. Bi-directional transport assays or concentration equilibrium transport assays (CETA) were performed by using MDR1-transfected and non-transfected cells to determine the substrate status of the following AEDs: phenytoin (PHT), phenobarbital (PB), ethosuximide (ESM), carbamazepine (CBZ), eslicarbazepine acetate (ESL), oxcarbazepine (OXC), (S)-licarbazepine (S-LC), carbamazepine-10,11-epoxide (CBZ-E), rufinamide (RFM), lacosamide (LCM), zonisamide (ZNS), and pregabalin (PGB). LLC-PK1 cells transfected with MDR1 variants were used to evaluate the effects of MDR1 polymorphisms on transport activity of AEDs in CETA. / Purpose: Epilepsy is a major neurological disorder, affecting more than 50 million people worldwide. Antiepileptic drugs (AEDs) do not effectively treat 30--40% of patients. Export of AEDs by P-glycoprotein (Pgp, ABCB1, or MDR1), which is overexpressed in the blood-brain barrier in drug-resistant patients, may be a mechanism for resistance to AEDs. Single nucleotide polymorphisms (SNPs) 1236C>T, 2677G>T and 3435C>T have been associated with drug-resistant epilepsy and were sometimes found to have effects on Pgp activities. But whether (or which) AEDs are transported by Pgp remains unclear, and there is no direct evidence showing that polymorphisms affect the transport of AEDs by Pgp. Therefore, we propose to use monolayers of cells transfected with the MDR1 variants to investigate 1) which AEDs are substrates for Pgp; and 2) the effect of MDR1 polymorphisms (1236C>T, 2677G>T, and 3435C>T) on AED transport. / Results: In CETA, PHT, PB, and LCM were transported by MDR1-transfected cells from basolateral to apical sides, while RFM, ZNS, PGB and ESM were not transported. Pgp did not transport CBZ, but did transport its active metabolite CBZ-E. Pgp also pumped ESL, OXC, and their active metabolite S-LC. The transport of these drugs can be completely blocked by Pgp inhibitor verapamil or tariquidar. In bi-directional transport assays, the Papp for the basolateral to apical direction in MDR1-transfected cells was significantly higher than in non-transfected cells for PHT, OXC, ESL, and S-LC, and not for PB, CBZ-E, CBZ, or ESM. / To compare the extent of basolateral-to-apical transport efficiency of different variants, we calculated the amount of the transported drugs divided by expression level of MDR1 in the apical chamber for each variant. In the G418 selection condition, compared with reference haplotype CGC, the CTC haplotype increased Pgp activity to transport OXC and ESL, while the CGT and CTT haplotypes did not significantly affect Pgp function. In the vincristine sulfate selection condition, compared with CGC, the haplotype CTT decreased Pgp activity, while other haplotypes, including CGC, CGT, CAC, CTC, TGC, TGT, TTT, and TTC, did not affect function. Selection by vincristine sulfate may raise expression of Pgp and eliminate differences among the variants. / Zhang, Chunbo. / Advisers: Vincent Hon Leung Lee; Lawrence William Baum; Zhong Zuo; Patrick Kwok Leung Kwan. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 192-221). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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Phleboviruses are emerging zoonotic pathogens which constitute a global threat to human and animal health. The mosquito-borne Rift Valley fever virus (RVFV) is a widespread problem across the African continent and causes regular deadly outbreaks in ruminants. The recently emerged severe fever with thrombocytopenia syndrome virus (SFTSV) is a serious human public health concern in China which has rapidly spread to Japan and Korea with fatality rates as high as 16-30%. Phleboviral cell entry is mediated by two viral glycoproteins: the class II fusion protein Gc and the lesser known Gn. Initial cell attachment is glycan dependent and the penetration into the cell cytoplasm is mediated by the Gc fusion protein which catalyses viral and cell membrane merger. The entry mechanism is not well understood from a structural perspective which limits mechanistic insights. The purpose of this thesis is to further our understanding of the cell entry process by filling in the missing structural information on the phleboviral glycoprotein layer. To this end, an integrated structural approach using cryo-EM and X-ray crystallography was adopted. The crystal structure of the Gn ectodomain is presented which reveals an unprecedented structural relationship with seemingly unrelated viruses. Single-particle cryo-EM and localized reconstruction reveal the glycoprotein layer of the RVFV and a pseudo-atomic model of the RVFV is presented. The assembly shows the shielding of the Gc fusion protein and suggests that the Gn functions as a fusion chaperone. The post-fusion crystal structure of the Gc protein from SFTSV further consolidates a mechanism of membrane fusion by class II fusion proteins. Finally, preliminary data on receptor binding and mechanism of antibody mediated neutralization are presented. The work presented herein provides a novel platform for studying and understanding entry and assembly of phleboviruses as well as the design of novel therapeutics.

Oviductin is an oviduct-specific and high-molecular-weight glycoprotein that has been suggested to play important roles in the early events of reproduction. The present study was undertaken to localize the oviductin binding sites in the uterine epithelial cells of the golden hamster (Mesocricetus auratus) both in situ and in vitro, and to detect a hamster oviductin homologue in the female rat reproductive tract. Immunohistochemical localization of oviductin in the hamster uterus revealed certain uterine epithelial cells reactive to the monoclonal anti-hamster oviductin antibody. In order to study the interaction between hamster oviductin and the endometrium in vitro, a method for culturing primary hamster uterine epithelial cells has been established and optimized. Study with confocal microscopy of the cell culture system showed a labeling pattern similar to what was observed using immunohistochemistry. Pre-embedding immunolabeling of cultured uterine epithelial cells also showed gold particles associated with the plasma membrane and microvilli. These results demonstrated that hamster oviductin can bind to the plasma membrane of certain hamster uterine epithelial cells, suggesting the presence of a putative oviductin receptor on the uterine epithelial cell surface. In the second part of the present study, using the monoclonal anti-hamster oviductin antibody that cross-reacts with the rat tissue, we have been able to detect an oviduct-specific glycoprotein, with a molecular weight of 180~300kDa, in the female rat reproductive tract. Immunohistochemical labeling of the female rat reproductive tract revealed a strong immunolabeling in the non-ciliated oviductal epithelial cells and a faint immunoreaction on the cell surface of some uterine epithelial cells. Ultrastructurally, immunogold labeling was restricted to the secretory granules, Golgi apparatus, and microvilli of the non-ciliated secretory cells of the oviduct. In the uterus, immunogold labeling was observed on the cell surface of some uterine epithelial cells. Furthermore, electron micrographs of ovulated oocytes showed an intense immunolabeling for rat oviductin within the perivitelline space surrounding the ovulated oocytes. The findings of the present study demonstrated that oviductin is present in the rat oviduct and uterus, and it appears that, in the rat, oviductin is secreted by the non-ciliated secretory cells of the oviduct. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2008-02-28 10:26:46.836 / This work was sponsored by Canadian Institutes of Health Research

The importance of arenavirus glycoprotein processing has only been understood within the past decade, with the majority of work focused on the Old World arenaviruses. Evidence has shown that SKI-1/S1P (subtilisin kexin isozyme-1/site 1 protease) is the cellular protease responsible for glycoprotein cleavage in Old and New World arenaviruses. Furthermore, glycoprotein cleavage is shown to be necessary for the production of infectious virus particles in Lassa and Junín viruses. In this thesis, evidence is provided that the recently emerged Chapare virus (New World) is also processed by SKI-1/S1P. Additionally, novel serpin-based SKI-1 inhibitors were shown to effectively prevent SKI-1 mediated cleavage. Using a wide panel of recombinant vesicular stomatitis viruses expressing New World arenavirus glycoproteins, these inhibitors were capable of significantly reducing viral titres. This provides strong evidence that SKI-1 inhibitors can be used as an effective treatment against the majority of New World Clade B arenaviruses and LASV in vivo.

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