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Investigation of the Role of the Glycoprotein Pentamer Complex in the Pathogenesis of Human Cytomegalovirus Infection
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Investigation of the Role of the Glycoprotein Pentamer Complex in the Pathogenesis of Human Cytomegalovirus Infection
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Investigation of the Role of the Glycoprotein Pentamer Complex in the Pathogenesis of Human Cytomegalovirus Infection
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Dissertation
Investigation of the Role of the Glycoprotein Pentamer Complex in the Pathogenesis of Human Cytomegalovirus Infection
2024
Overview
Human Cytomegalovirus (HCMV) infection is a significant public health concern, with congenital HCMV (cHCMV) infection affecting approximately 1% of all live births. Of these infected infants, 10-15% develop symptomatic disease with complications including microcephaly, neurodevelopmental delay, and sensorineural hearing loss (SNHL). However, the long-term consequences of cHCMV infection are variable, with an additional 10-15% of asymptomatic infants developing SNHL later in life. The underlying mechanisms driving these diverse outcomes could be multifactorial but remain unknown. One potential factor contributing to these variations is the diversity of glycoproteins on HCMV envelope. It has been suggested that differences in glycoprotein genotypes may influence the virus's virulence. The HCMV virion is a complex structure, and the pentamer complex, essential for viral entry and replication in various cells, is a key component. The UL128 gene encodes a protein integral to this complex. This study aimed to investigate the role of UL128 in HCMV pathogenesis and to identify novel viral and host factors involved in cHCMV infection. To investigate the role of UL128, three UL128 variants derived from published HCMV whole genome isolates were introduced into a recombinant HCMV BAC (RCMV111) using a two-step Red-mediated recombination system. The resulting recombinant viruses were characterised phenotypically through infectivity assays, cytokine response, and glycosylation patterns via enzyme-linked lectin assay analysis. For transcriptional analysis, published RNA sequencing HCMV infected monocytes data was used to identify differentially expressed genes and gene fusion. Furthermore, selected in-silico predicted gene fusions were experimentally tested in MRC-5 and ARPE-19 cells. Recombinant HCMV strains expressing the different UL128 variants successfully regained tropism for epithelial cells. However, these viruses exhibited no significant phenotypic differences compared to the parental strain in cytokine response and lectin assays, while infectivity showed decreased infectivity in fibroblasts compared to the wild type. RNA sequencing of HCMV infected monocytes revealed differential expression of several genes, including EPHB2. Further analysis demonstrated a similar pattern of EPHB2 gene expression in HCMV infection of MRC-5 cells. Experimental validation of in-silico predicted gene fusions yielded variable results in MRC-5 and ARPE-19 cells. This study provides new insights into the complex nature of HCMV pathogenesis. While UL128 is essential for viral tropism, our findings suggest that the specific UL128 variant does not significantly impact viral phenotype in the RCMV1111 model. The identification of differentially expressed genes and gene fusions in HCMV infected monocytes highlights the potential of transcriptomic analysis for uncovering novel viral and host factors involved in infection. Further investigation of these factors is warranted to understand their precise roles in HCMV pathogenesis.
