Endoplasmic Reticulum (ER) Reorganization and Intracellular Retention of CD58 Are Functionally Independent Properties of the Human Cytomegalovirus ER-Resident Glycoprotein UL148

Abstract

The human cytomegalovirus (HCMV) endoplasmic reticulum (ER)-resident glycoprotein UL148 is posited to play roles in immune evasion and regulation of viral cell tropism. UL148 prevents cell surface presentation of the immune cell costimulatory ligand CD58 while promoting maturation and virion incorporation of glycoprotein O, a receptor binding subunit for an envelope glycoprotein complex involved in entry. Meanwhile, UL148 activates the unfolded protein response (UPR) and causes large-scale reorganization of the ER. In order to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated six charged cluster-to-alanine (CCTA) mutants within the UL148 ectodomain and compared them to wild-type UL148, both in the context of infection studies using recombinant viruses and in ectopic expression experiments, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79 to 83 (CC3) and 133 to 136 (CC4), retained the potential to impede CD58 surface presentation. Of the six mutants, only CC3 retained the capacity to reorganize the ER, but it showed a partial phenotype. Wild-type UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which implies a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses surface presentation of CD58 but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate various degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that the capacity to cause ER reorganization is unique to HCMV UL148. IMPORTANCE In myriad examples, viral gene products cause striking effects on cells, such as activation of stress responses. It can be challenging to decipher how such effects contribute to the biological roles of the proteins. The HCMV glycoprotein UL148 retains CD58 within the ER, thereby preventing it from reaching the cell surface, where it functions to stimulate cell-mediated antiviral responses. Intriguingly, UL148 also triggers the formation of large, ER-derived membranous structures and activates the UPR, a set of signaling pathways involved in adaptation to ER stress. We demonstrate that the potential of UL148 to reorganize the ER and to retain CD58 are separable by mutagenesis and, possibly, by evolution, since chimpanzee cytomegalovirus UL148 retains CD58 but does not remodel the ER. Our findings imply that ER reorganization contributes to other roles of UL148, such as modulation of alternative viral glycoprotein complexes that govern the virus’ ability to infect different cell types.

The human cytomegalovirus protein UL116 interacts with the viral ER resident glycoprotein UL148 and promotes the incorporation of gH/gL complexes into virions

“…New recombinant viruses were constructed by en passant BAC recombineering (30,31), as previously described (6,18,(32)(33)(34). Briefly, for each recombinant virus, a primer pair (see below and Table 1) is used to PCR amplify an I-SceI-AphAI cassette from a BAC DNA template that contains the cassette.…”

Engineering, Decoding and Systems-Level Characterization of Chimpanzee Cytomegalovirus

“…This latter group includes UL148 which has early expression kinetics in CCMV (TP2) but late kinetics (TP5) in HCMV infection. HCMV-UL148 has two distinct functions: it promotes the intracellular retention of CD58, thereby impeding the detection of infected cells by the immune system (Wang et al, 2018); and it causes large-scale remodeling of the endoplasmic reticulum (ER), thereby protecting the viral glycoprotein O (UL74) from ER-associated degradation (Nguyen et al, 2020(Nguyen et al, , 2018. Since only the immune evasion but not the ER remodeling function is conserved in CCMV-UL148 (Nguyen et al, 2020), it is conceivable that its earlier expression reflects the lack of ER remodeling function.…”

Engineering, decoding and systems-level characterization of chimpanzee cytomegalovirus

“…This latter group includes UL148 which has early expression kinetics in CCMV (TP2) but late kinetics (TP5) in HCMV infection. HCMV-UL148 has two distinct functions: it promotes the intracellular retention of CD58, thereby impeding the detection of infected cells by the immune system [74]; and it causes large-scale remodeling of the endoplasmic reticulum (ER), thereby protecting the viral glycoprotein O (UL74) from ER-associated degradation [75,76]. Since only the immune evasion but not the ER remodeling function is conserved in CCMV-UL148 [76], it is conceivable that its earlier expression reflects the lack of ER remodeling function.…”