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Roles of the UL148 glycoprotein in human cytomegalovirus cell tropism

UL148 糖蛋白在人巨细胞病毒细胞趋向性中的作用

基本信息

批准号：
9029924
负责人：
Jeremy Phillip Kamil
金额：
$ 35.68万
依托单位：
LOUISIANA STATE UNIV HSC SHREVEPORT
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-15 至 2020-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9029924
关键词：
Address Affect Amino Acids Bacterial Artificial Chromosomes Binding Biological Biological Process Cell membrane Cells Complex Congenital Abnormality Cysteine Cytomegalovirus Cytomegalovirus Vaccines Cytoplasmic Tail Data Defect Disease Disulfides Ectopic Expression Employee Strikes Endoplasmic Reticulum Endothelial Cells Epithelial Epithelial Cells Escherichia coli Exhibits Fibroblasts Genes Genetic Polymorphism Genome Glycoproteins Goals Herpesviridae Human Human Cloning Human Herpesvirus 4 Human Herpesvirus 6 Human body Immune system Immunocompromised Host Immunoprecipitation Individual Infection Integral Membrane Protein L Forms Label Laboratories Leukocytes Life Link Mass Spectrum Analysis Measures Mediator of activation protein Molecular Newborn Infant Opportunistic Infections Pathogenesis Physiologic pulse Play Proteins Public Health Research Project Grants Role Site Techniques Tropism Variant Viral Viral Genes Viral Proteins Virion Virus Western Blotting base cell type dimer disulfide bond in vivo insight mutant pathogen protein complex protein protein interaction public health relevance recombinant virus restoration tool

项目摘要

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a herpesvirus pathogen that causes life threatening infections in people with compromised immune systems, and congenital defects in newborn children. HCMV depends on its viral envelope glycoproteins to infect human cells. A pair of viral glycoproteins, gH and gL, form a complex, gH/gL, that plays crucial roles in entry of HCMV. As is also the case for Epstein Barr virus (EBV) and human herpesvirus 6 (HHV-6), HCMV virions contain two alternative forms of gH/gL. In HCMV, one of these is a complex with gO, gH/gL/gO, and the other is a complex with UL128, UL130 and UL131, gH/gL/UL128-131. The gH/gL/gO complex suffices for infection of fibroblasts, while gH/gL/UL128-131 is necessary for infection of epithelial and endothelial cells. Viral factors that regulate the relative abundance of alternative gH/gL complexes have not been previously described for any herpesvirus. Intriguingly, we have discovered that HCMV encodes an ER-resident glycoprotein, UL148, which may represent the first example of such a factor. UL148 strongly influences the relative amounts of the two gH/gL complexes on virions, and has large effects on cell tropism. The Specific Aims of this project are to define the influence of UL148 on cell tropism and on maturation of gH/gL complexes, and to identify and characterize protein-protein interactions involving UL148. To carry out these aims, UL148 null mutants of multiple HCMV strains will be characterized, and whether variation in the UL148 gene contributes to the well-documented differences in tropism between HCMV strains will be determined. Studies will also be conducted to determine the effects on tropism and gH/gL when the UL148 is restored to laboratory-adapted HCMV strain AD169, which otherwise lacks the gene. Proteins that co-purify with UL148 during immunoprecipitation from infected cells will be identified by Western blotting and mass spectrometry. Protein interactions involving UL148 and its effects on maturation of gH/gL complexes will be characterized in uninfected cells ectopically expressing the proteins. Chimeric UL148 proteins, in which specific domains of UL148 are swapped with topologically similar proteins, and mutant UL148 proteins, lacking either its ectodomain or cytoplasmic tail, will be used to define the domains necessary for its functions and interactions. Recombinant viruses will be constructed using infectious bacterial artificial chromosome clones of HCMV and sequence manipulation techniques in E. coli. Western blotting, pulse-chase labeling, and enzymatic approaches will be used to visualize the influence of UL148 on gH/gL complexes. Cell tropism will be measured by comparing infectivity on epithelial cells, endothelial cells and fibroblasts. Completion of the project will result in new information that addresses gaps in the current understanding of how HCMV cell tropism is regulated. The resulting findings may have implications for HCMV vaccines, and for understanding the cell tropism of other medically important herpesviruses, such as HHV-6 and EBV, which likewise deploy alternative gH/gL complexes.