Roles of the UL148 glycoprotein in human cytomegalovirus infection

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This project focuses on the role of glycobiology in human cytomegalovirus (HCMV) immune evasion strategies. Our long-term goal is to understand how the virus exploits host protein glycosylation pathways to shield its virions and decorate the surfaces of infected cells with oligosaccharide patterns that prevent antibody binding and/or suppress immune responses. The Specific Aims are (1) to evaluate how the HCMV ER resident protein UL148 impacts the glycocalyx, or “sugar cloak,” of virions and infected cells, (2) to carry out a structure function analysis of UL148 to determine the mechanisms by which UL148 activates sculpts the host cell to enhance glycosylation of virion envelope proteins, and (3) to evaluate how UL148 contributes to evasion of antibody responses. Throughout the three Specific Aims, wild-type (WT) and UL148-null mutant viruses are evaluated in comparison to each other. In Specific Aim 1, mass spectrometry based ‘glycomics’ is applied to profile N-linked oligosaccharide structures conjugated to virion envelope proteins and surface proteins of infected cells in the presence versus absence of UL148. A second aspect of Aim 1 makes use of mass spectrometry-based proteomics together with genetic and pharmacological perturbations targeting the unfolded protein response (UPR) and cellular genes whose expression is positively regulated by UL148 to determine how the viral protein harnesses the host cell’s stress response circuitry toward the generation of virions that resist neutralization by antibodies. In Specific Aim 2, computational predictions of UL148’s three-dimensional protein structure are used to guide mutagenesis studies aimed at understanding the molecular basis for its biological functions, focusing in large part on the roles of (i) a consensus metal binding motif and (ii) two non- conserved cysteine residues. Aim 2 also leverages structural models non-human primate cytomegaloviruses homologs to elucidate the determinants of HCMV UL148’s unique biological functions. These studies will make use of recombinant HCMVs to test the impact of selected mutations and chimeric UL148 proteins in the context of infected cells. In Specific Aim 3, we will (i) characterize UL148’s impact on neutralization resistance of virions while targeting glycan modification enzymes hypothesized to be important for biogenesis of neutralization resistant virions, (ii) map specific antigenic domains on virion glycoproteins whose reactivity to neutralizing antibodies is impacted by UL148, (iii) compare the humoral immunogenicity of UL148 competent versus UL148-null HCMV virions in a mouse model, testing for neutralizing antibody titers, and overall polyclonal antibody titers against whole virions, viral envelope proteins and internal virion antigens. Collectively, these studies will address a key knowledge gap concerning how enveloped large DNA viruses exploit glycobiology to evade adaptive immunity and establish persistent infection.