Browsing by Author "Schafer, Georgia"

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    Characterisation of human surfactant protein A and recombinant human vimentin in their modulation of HPV16 pseudovirus infection
    (2019) Carse, Sinead; Schafer, Georgia; Katz, Arieh
    Infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, where low-and middle-income countries (LMIC) have the highest incidence. Prophylactic HPV vaccines exist but LMIC have limited access. Therefore, alternative preventative measures against HPV infection and cervical cancer progression are needed. Two human proteins have been identified in our laboratory that modulate HPV16 pseudovirus (HPV16-PsVs) infection in vitro, namely surfactant protein A (SP-A) and recombinant human vimentin (rhVim). Previous work suggested SP-A mediated immune recognition of HPV since SP-A-coated HPV16- PsVs enhanced viral uptake by RAW264.7 murine macrophages. These initial observations were confirmed using a murine C57BL/6 cervicovaginal challenge model: pre-incubation of HPV16- PsVs with purified human SP-A significantly reduced the level of HPV16-PsV infection in vivo. Moreover, when isolated cells from female reproductive tracts of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in viral uptake by eosinophils, neutrophils, monocytes and macrophages were observed over time using SP-A-pre-coated virions compared to control particles. Compared to SP-A mediated modulation of HPV infection through activation of innate immune responses, rhVim was suggested to directly interfere with HPV entry into host cells. Indeed, supplementation with non-filamentous rhVim resulted in decreased viral uptake by NIKS cells which was confirmed in vivo using the murine C57BL/6 cervicovaginal HPV16-PsVs challenge model. Co-localisation analysis employing confocal imaging, revealed that rhVim-coated HPV16- PsVs co-localised, to a lesser degree, with surface-expressed heparan sulphate proteoglycans (HSPGs) than control particles. Removal of surface HSPGs on NIKS cells decreased HPV16-PsVs cell surface binding and internalisation, while pre-incubation of HPV16-PsVs with rhVim decreased viral particle binding and internalisation to a greater extent. This indicates that rhVim may modulate HPV16 infection by interfering with its attachment to HSPGs as well as viral engagement with the yet unknown entry receptor(s). In summary, both SP-A and vimentin modulate HPV16-PsVs infection by different mechanisms. These in vivo studies strongly confirm previous in vitro observations, rendering both proteins potentially suitable for further development into possible candidates for use in topical microbicides, which may provide protection against new HPV infections.
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    Characterisation of Kaposi's sarcoma-associated herpesvirus (KSHV)-driven pathology and disease outcome in HIV infected South African patients
    (2020) Blumenthal, Melissa; Schafer, Georgia; Katz, Arieh
    Kaposi's sarcoma-associated herpesvirus (KSHV), a gamma-herpesvirus with a particularly high seroprevalence in Sub-Saharan Africa (SSA), is the etiological agent of the endothelial tumour Kaposi's sarcoma (KS), the most common acquired immunodeficiency syndrome (AIDS)-related malignancy worldwide and particularly in SSA. It also causes primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and KSHV inflammatory cytokine syndrome (KICS). AIDS-related deaths have declined, due to global scale-up of antiretroviral therapy (ART). However, the vast majority of these occurred in SSA, where tuberculosis (TB) is the leading cause of mortality among human immunodeficiency virus (HIV)-infected individuals, accounting for a third of all AIDS-related deaths. The exceptionally high burden of suspected TB in SSA causes misdiagnosis or delayed diagnosis of diseases mimicking TB, such as several pathologies associated with KSHV. KSHV infection is essential but insufficient for the development of KS and other KSHV-associated pathologies; precipitating factors, such as HIV-related immune suppression and potentially genetic predisposition, are required. The erythropoietin-producing hepatocellular carcinoma (Eph) receptor A2 protein (EPHA2) tyrosine kinase receptor is a promising candidate for studies on genetic variants as it potentially acts on two levels: susceptibility to KSHV infection (being one of the key receptors utilised by KSHV for cell entry and intracellular trafficking) and susceptibility to KS development (being implicated in oncogenesis). Despite the high seroprevalence in SSA, the contribution of dysregulated KSHV lytic replication or host KSHV receptor variations to disease outcome in HIV-infected patients is unknown. We hypothesised that KSHV lytic reactivation plays yet unrecognised roles for morbidity and mortality in high HIV settings and to this end, we conducted a cohort study of 682 HIV-positive critically ill patients admitted to Khayelitsha Day Hospital, South Africa, investigated for TB, and followed for 12-weeks to ascertain vital status. We demonstrated that elevated blood KSHV viral load (VL) was a strong predictor of death in hospitalised HIV-infected patients without microbiologically proven TB. Further, we identified and validated variants in the EPHA2 protein tyrosine kinase and sterile alpha motif domains that were significantly associated with susceptibility to infection, KS development and/or KSHV VL in 300 South African HIV-infected patients, by aggregate by-gene analysis. In order to elucidate the functional significance of the identified EPHA2 missense mutations, we knocked out endogenous EPHA2 by CRISPR/Cas9 in the human endothelial cell line, HuARLT2, and reintroduced the wild type and mutant EPHA2 open reading frames by lentiviral transduction. These engineered cells were assessed for baseline EPHA2 phosphorylation levels and susceptibility to KSHV infection utilising recombinant KSHV in binding, internalisation and infection assays. We found that the EPHA2 mutant c.2254T>C (p.Leu700Pro) in the tyrosine kinase domain, associated with KS in our patient cohort, was deficient in tyrosine phosphorylation and less permissive to rKSHV infection when introduced as a single mutation or as a double mutant together with c.2257A>C (p.Asp701Ala) which was found to be in linkage disequilibrium with it. Another tyrosine kinase domain variant, c.2688G>S (p.Ala845Pro), found to be overrepresented among KS patients, had enhanced baseline tyrosine phosphorylation levels. These findings validated the patient-derived data on the molecular level by assigning functional consequences to some mutants which might have implications for the development of future biomarkers predicting KS susceptibility in high-risk populations. In summary, this novel research contributes to the understanding of KSHV-associated pathology and disease outcome. It identified KSHV VL as a potential biomarker to predict KSHV-associated diseases and mortality and assessed the contribution of KSHV entry receptor EPHA2 variations to KSHV-associated pathologies, with potential clinical implications, by facilitating the development of novel diagnostic and surveillance tools.
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    Cryo-electron microscopy of HPV16 pseudovirions reveal changes in capsid conformation upon furin cleavage
    (2021) Marx, Melissa Lauren; Schafer, Georgia; Woodward, Jeremy
    Persistent infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, a leading cause of cancer deaths in women worldwide. There are no treatments for HPV infection, and although prophylactic vaccines are effective and safe, they are HPV type specific, provide little therapeutic benefit and developing countries often have limited access to these. Therefore, additional measures against HPV infection are urgently needed. Preventing HPV entry into host cells is an attractive option for therapeutic intervention. The HPV capsid is icosahedral and consists of two proteins, L1 and L2, which participate in entry and infection of host cells. During entry, the virus capsid attaches to the cell surface via binding to heparan sulphate proteoglycans (HSPGs). Cleavage of L2 by a host protease, furin, is necessary for infection and is thought to facilitate a conformational change in the virus capsid. Furin cleavage may affect the ability of HPV to bind to sulphated glycoproteins and a HSPG substitute, heparin. Understanding these proposed structural changes may aid in the development of therapeutics targeting virus entry. Here, we directly visualize the conformation changes to HPV16 pseudovirions (HPV16 PsVs) resulting from cleavage of L2 by exogenous furin using cryoelectron microscopy (cryo-EM). At 5 Å resolution, we observed that furin-cleaved HPV16 PsVs capsids display widespread changes in the arrangement of capsomeres relative to uncleaved control virions. This structural change is relevant because heparin has previously been observed to bind to the HPV16 capsid in the canyon surrounding the capsomere at the five-fold icosahedral symmetry axis, but not in other canyons between capsomeres, related by pseudo-symmetry. This suggests that differences in the relative orientations of the surrounding capsomeres to each other either prevent or allow heparin binding. We observed a narrowing of the putative heparin binding site by 0.4 Å after furin cleavage and propose that this change may be responsible for the transfer of HPV from cell-surface HSPGs to the unknown entry receptor(s) by a yet unidentified mechanism.
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    Systematic Kaposi-Sarcoma Herpesvirus (KSHV) genome analysis from a South African HIV- and KSHV-positive patient cohort
    (2024) Thomas, Melissa; Schafer, Georgia
    Kaposi sarcoma herpesvirus (KSHV) is an oncogenic virus and the etiological agent of Kaposi's sarcoma, the most common AIDS-associated cancer. KSHV infection occurs primarily in Sub-Saharan Africa (SSA) where it is also associated with other pathologies such as Multicentric Castleman's Disease, Primary Effusion Lymphoma and KSHV-associated inflammatory cytokine syndrome, which also occur primarily with HIV co-infection. IN SSA most individuals ac-quire KSHV infection during childhood where it remains undetected until later in life when the burden of HIV takes its effect on the immune system. There are 6 predominant KSHV subtypes world-wide, namely A, B, C, D, E and F which are determined by the highly variable K1 region of the KSHV genome. Sequencing and characterizing the circulating subtypes in a specific geographical region assist in tracking evolutionary changes as well as malignant outcomes associated with different subtypes. The aim of this study was to determine the circulating KSHV subtypes in the Western Cape region of South Africa. A total of 57 DNA samples isolated from peripheral blood of confirmed HIV/KSHV co-infected patients were selected according to KSHV viral load (VL) (> 5 copies/10 μL) and volume (> 5 μL). Of these, 20 were successfully Sanger sequenced to determine K1 subtype. Additionally, 9 samples met the criteria for whole-genome sequencing using Next Generation Sequencing (NGS). This study produced 29 K1 sequences (20 via Sanger and 9 via NGS) of which 26 were of the A subtype, specifically A5, and the remaining 3 were of the B subtype, namely B2. These results are consistent with previous studies from the same region where a trend of high A5 and B subtypes in AIDS-KS patients were reported. ORF-K15 is another variable and lytic gene associated with KSHV subtyping (alleles M, N and P). Sequencing results of the K15 gene, revealed two P subtypes, three M subtypes and four N subtypes in our samples. These novel results from the Western Cape of South Africa point towards an interesting distribution of subtypes contributing to previous reports of K15 P and M subtypes being the most prevalent in Africa. There are limited whole-genome sequences available for South Africa, therefore these sequences provide a significant contribution to the pool of sequences available for this region.
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    Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner
    (BioMed Central Ltd, 2013) van Rooyen, Beverley; Schafer, Georgia; Leaner, Virna; Parker, M
    BACKGROUND: Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells. RESULTS: We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells. CONCLUSION: We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.