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Characterization of the Influences of Human Cytomegalovirus University of Montana ScholarWorks at University of Montana Graduate Student Theses, Dissertations, & Professional Papers Graduate School 2020 Characterization of the Influences of Human Cytomegalovirus Glycoprotein O (gO) Expression on gH/gL Complexes Assembly and Its Polymorphisms on Cell-free and Cell-to-cell Spread, and Antibody Neutralization. Le Z. Day Follow this and additional works at: https://scholarworks.umt.edu/etd Part of the Biochemistry Commons, Cell Biology Commons, Molecular Biology Commons, and the Virology Commons Let us know how access to this document benefits ou.y Recommended Citation Day, Le Z., "Characterization of the Influences of Human Cytomegalovirus Glycoprotein O (gO) Expression on gH/gL Complexes Assembly and Its Polymorphisms on Cell-free and Cell-to-cell Spread, and Antibody Neutralization." (2020). Graduate Student Theses, Dissertations, & Professional Papers. 11543. https://scholarworks.umt.edu/etd/11543 This Dissertation is brought to you for free and open access by the Graduate School at ScholarWorks at University of Montana. It has been accepted for inclusion in Graduate Student Theses, Dissertations, & Professional Papers by an authorized administrator of ScholarWorks at University of Montana. For more information, please contact [email protected]. 2020 Characterization of the influences of Human Cytomegalovirus glycoprotein O (gO) expression on gH/gL complexes assembly and its polymorphisms on cell-free and cell-to-cell spread, and antibody neutralization. Le Zhang Day Characterization of the influences of Human Cytomegalovirus glycoprotein O (gO) expression on gH/gL complexes assembly and its polymorphisms on cell-free and cell-to-cell spread, and antibody neutralization. By Le Z. Day B.S., Anhui University, Hefei, Anhui, China, 2013 Dissertation presented in partial fulfillment of the requirements for the degree of Ph.D. in Biochemistry and Biophysics The University of Montana Missoula, MT May 2020 Approved by: Scott Whittenburg, Dean of The Graduate School Graduate School Dr. Brent Ryckman, Research Advisor Division of Biological Sciences Dr. Ekaterina Voronina Division of Biological Sciences Dr. J. Stephen Lodmell Division of Biological Sciences Dr. Bruce Bowler Department of Chemistry and Biochemistry Dr. Mark Grimes Division of Biological Sciences II Day, Le, Ph.D., May 2020 Biochemistry and Biophysics Abstract Title: Characterization of the influences of Human Cytomegalovirus glycoprotein O (gO) expression on gH/gL complexes assembly and its polymorphisms on cell-free and cell-to-cell spread, and antibody neutralization. Research Advisor: Dr. Brent J. Ryckman Human cytomegalovirus (HCMV) is widely spread throughout the world and immunocompromised individuals can suffer severe diseases from HCMV infection. Once the infection is established, HCMV can spread through the body and infect many major somatic cell types. The glycoproteins H and L (gH/gL) on HCMV envelope can be bound by either gO or the UL128-131 proteins to form complexes gH/gL/gO and gH/gL/UL128-131 that are critical for viral entry and spread, and these two complexes are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131. gO is one of the most diverse loci among strains with 10-30% of amino acid sequence differences. In this thesis I explored the mechanisms behind the complex assembly differences between strains and the impacts of interstrain gO diversity on the biology of HCMV. My results uncovered that the strain variations in the assembly of gH/gL complexes is due to the differences in the expression level of gO and UL128-131, while gO amino acid sequence differences have no influence on the complexes assembly. Interestingly, the diversity of gO has dramatic impacts on HCMV cell-free and cell-to-cell spread as well as on antibody neutralization and these effects of gO polymorphisms are epistatically dependent on other variable loci in the virus genome. My study could help to understand the complexity of genotypes observed in clinical samples and decode the challenge for intervention approaches against HCMV. III Table of Contents Table of Contents ……………………………………………………………………………………… IV List of Figures ..………………………………………………………………………………………… VI List of Tables ……………………………………………………………………………………………. VIII Acknowledgements……………………………………………………………………………………..IX Chapter I. Introduction……………………………………………………………………………… . 1 Overview of Human Herpesviruses………………………………………………………… 2 Herpesviridae …………………………………………………………………………… 2 Human Herpesviruses ……………………………………………………………….. 2 Overview of Human Cytomegalovirus ………………………………………………….. 4 HCMV structure…………………………………………………………………………… 4 HCMV lifecycle…………………………………………………………………………… 7 HCMV genetic variability…………………………………………………………….. 10 HCMV immune evasion……………………………………………………………….. 12 HCMV pathology…………………………………………………………………………. 13 HCMV treatment and prevention…………………………………………………. 15 Focus of the dissertation………………………………………………………………………… 18 Chapter II. Expression levels of glycoprotein O (gO) vary between strains of Human Cytomegalovirus, influencing the assembly of gH/gL complexes and virion infectivity…………………………………………………………………………………………. 21 Introduction………………………………………………………………………………………….. 22 Results………………………………………………………………………………………………….. 24 IV Discussion .………………………………………………………………………………………….. 39 Materials and methods ………………………………………………………………………… 44 Chapter III. Polymorphisms in Human Cytomeglaovirus gO exert epistatic influences on cell-free and cell-to-cell spread, and antibody neutralization on gH epitopes………………………………………………………………………………………………….. 51 Introduction…………………………………………………………………………………………... 52 Results…………………………………………………………………………………………………... 56 Discussion……………………………………………………………………………………………… 80 Materials and methods .……………………………………………………………………… 89 Chapter IV. Conclusion, discussion, and future directions………………………… 96 References………………………………………………………………………………………………. 123 V List of Figures Fig 1.1 Particle in cytoplasm of CMV-infected cells………………………………………………. 4 Fig 1.2 HCMV genome structure………………………………………………………………………….. 5 Fig 2.1 Comparison of protein expression between TR and ME……………………………26 Fig 2.2 Analysis of ER-to-trans-Golgi compartment trafficking of glycoproteins in TR- or ME-infected cells………………………………………………………………………………………….. 27 Fig 2.3 Specific infectivity of parental and TR-ME heterologous gO recombinants...30 Fig 2.4 Immunoblot analysis of gH/gL complexes in parental viruses and TR-ME heterologous gO recombinants………………………………………………………………………… 30 Fig 2.5 Immunoblot analysis of gO expression in cells infected with parental viruses and TR-ME heterologous gO recombinants…………………………………………………………32 Fig 2.6 Quantitative comparison of glycoprotein expression in TR- and ME-infected cells………………………………………………………………………………………………………………… 34 Fig 2.7 Analysis of glycoprotein turnover in TR- and ME-infected cells………………. 37 FIG 2.8 Ad vector overexpression of gO during ME replication…………………………… 38 FIG 2.9 Specific infectivity of ME virions produced under conditions of gO overexpression………………………………………………………………………………………………… 39 Fig 3.1 Immunoblot analysis of gH/gL complexes in parental and heterologous gO recombinant HCMV…………………………………………………………………………………………. 58 Fig 3.2 Relative fibroblast and epithelial cell tropism of parental and heterologous gO recombinant HCMV……………………………………………………………………………………. 61 VI Fig 3.3 Specific infectivity of parental and heterologous gO recombinant HCMV…. 63 Fig 3.4 Binding of parental and heterologous gO recombinant HCMV to fibroblasts……………………………………………………………………………………………………….. 66 Fig 3.5 Spread of parental and heterologous gO recombinant HCMV in fibroblast cultures…………………………………………………………………………………………………………… 71 Fig 3.6 Release of extracellular progeny by parental and heterologous gO recombinant HCMV in fibroblast cultures…………………………………………………………. 73 Fig 3.7 Spread of parental and heterologous gO recombinant HCMV in epithelial cell cultures…………………………………………………………………………………………………………… 75 Fig 3.8 Release of extracellular progeny by parental and heterologous gO recombinant HCMV ME in epithelial cell cultures…………………………………………….. 76 Fig 3.9 Neutralization of parental HCMV TR and heterologous gO recombinant by anti-gH antibodies…………………………………………………………………………………………... 79 Fig 3.10 Neutralization of parental HCMV MT and heterologous gO recombinant by anti-gH antibodies…………………………………………………………………………………………… 80 Fig 3.11 Association of gH and gO genotypes in 236 complete HCMV genome sequences in the NCBI database……………………………………………………………………… 88 Fig 4.1 Comparison of gO isoforms…………………………………………………………………… 99 VII List of Tables Table 1.1 Major properties of human herpesviruses…………………………………………….. 3 Table 2.1 Quantitative comparison of glycoprotein expression in TR- and ME- infected cells…………………………………………………………………………………………………….. 35 Table 2.2 Quantitative comparison of glycoprotein expression in TR_MEgO- and ME_TRgO-infected cells……………………………………………………………………………………. 36 Table 3.1 Immunoblot band density analyses of parental and heterologous gO recombinants…………………………………………………………………………………………………... 59 Table 3.2 Binding of parental and heterologous gO recombinant HCMV to fibroblasts………………………………………………………………………………………………………. 67 Table 3.3 Binding of parental and heterologous gO recombinant HCMV to epithelial cells………………………………………………………………………………………………………………… 68 VIII Acknowledgements I am sincerely grateful and thankful to my mentor Dr. Brent Ryckman for his training and guidance through my six years of graduate school. I remember the first thing Brent taught
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