Author

Li HeFollow

Defense Date

2021

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Michael A. McVoy

Second Advisor

Renfeng Li

Third Advisor

Zhao Lin

Fourth Advisor

Iain M. Morgan

Fifth Advisor

Daniel Nixon

Abstract

Human cytomegalovirus (HCMV) can be transmitted through body fluids and cause serious disease in the immunologically immature or immunocompromised population. HCMV entry utilizes different mechanisms involving various viral envelope glycoproteins. gB and gH/gL/gO (trimer) are involved in fibroblast entry and gH/gL/U128-131A (pentamer) is required in addition to trimer for epithelial and endothelial cell entry. gO is heavily glycosylated and highly polymorphic in different HCMV strains. gO in the parental TB40/E strain was completely or partially replaced by gOs from other HCMV strains. A quantitative luciferase-based assay demonstrated that certain polymorphic gOs from different strains can interfere with neutralization of fibroblast but not epithelial cell entry by monoclonal antibodies (mAbs) targeting epitopes in gH or gH/gL. Towne gO exhibited the most resistance to gH and gH/gL mAbs neutralization, whereas resistance conferred by other gO types was strain dependent. The N-terminal sequences of gO were responsible for the interference with mAb neutralization. gO polymorphisms did not influence the neutralizing activity of a gB mAb, hyperimmunoglobulin, or sera from HCMV-infected individuals. Even though HCMV entry into epithelial and endothelial cells shares a similar mechanism, significant titer differences in certain human sera suggested mechanistic differences in HCMV entry into these cell types.

HCMV entry and immediate-early (IE) protein expression in mucosal-derived epithelial cell lines occurred with similar efficiency as in ARPE-19 retinal pigment epithelial cells, but early and late protein expression, cell-to-cell spread, and HCMV DNA replication were dramatically restricted at a post-IE stage. Differentiation of mucosal epithelial cells did not significantly impact HCMV entry but profoundly reduced early and late protein expression. Clinical-like HCMVs UxcAp14 and ABV formed foci in human tonsil epithelial (HTE) cells, whereas UxcAp66 variant with US6 to U28 deletion did not, indicating a genetic determinant of viral spread in HTE cells. However, UxcAp14 could not spread on primary tonsil epithelial cells and while genetically authentic HCMVs shed in patient urines entered HTE cells efficiently, they failed to produce foci.

Neutralization by pentamer-specific mAbs and deletion of pentamer subunit genes revealed that HCMV can enter mucosal epithelial cells using both trimer complex dependent (TCD) and pentameric complex dependent (PCD) entry mechanisms and that TCD entry could be pentamer complex independent (PCI) while PCD entry could be trimer complex independent (TCI), but one of the two complexes were required. As previously reported, HCMV entry into fibroblasts was TCD/PCI while infection of ARPE-19 epithelial cells was TCD/PCD. In contrast, entry into HTE cells occurred frequently by a TCD/PCI mechanism and in normal oral keratinocyte (NOK), human adenoid epithelial (HAK), and keratinocyte from normal human epidermis (N/TERT) cells support for TCD/PCI entry was strongly induced by differentiation. By elucidating the region of amino acid changes in gO and cell lines from specific anatomic sites that contribute to HCMV resistance in antibody neutralization, it can promote the design of effective vaccines and incorporate molecular diagnostic tests in clinical laboratory to optimize immunotherapy.

Rights

© Li He

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

11-4-2021

Available for download on Tuesday, November 03, 2026

Included in

Viruses Commons

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