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 an effort to determine whether the seemingly disparate effects of UL148 are related or discrete, we generated charged-cluster-to-alanine (CCTA) mutants of six charged clusters within the UL148 ectodomain, and compared them against wildtype UL148, in the context of recombinant viruses and in ectopic expression, assaying for effects on ER remodeling and CD58 surface presentation. Two mutants, targeting charged clusters spanning residues 79-83 (CC3) and 133-136 (CC4), respectively, retained the potential to impede CD58 presentation, and did so to an extent comparable to wildtype. Of the six mutants, only CC3 retained the capacity to reorganize the ER, showing a partial phenotype. Wildtype UL148 accumulates in a detergent-insoluble form during infection. However, all six CCTA mutants were fully soluble, which may imply a relationship between insolubility and organelle remodeling. Additionally, we found that the chimpanzee cytomegalovirus UL148 homolog suppresses CD58 presentation but fails to reorganize the ER, while the homolog from rhesus cytomegalovirus shows neither activity. Collectively, our findings illustrate varying degrees of functional divergence between homologous primate cytomegalovirus immunevasins and suggest that ER reorganization is unique to HCMV UL148.IMPORTANCEIn 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 Nonstructural Glycoprotein UL148 Reorganizes the Endoplasmic Reticulum

“…Although intracellular forms of CD58 are, in our hands, refractory to detection by standard indirect immunofluorescence protocols (not shown), it seems reasonable to hypothesize that UL148 sequesters CD58 into the unusual ER structures that it induces. Nonetheless, recent work from our laboratory identified charged-cluster-to-alanine mutants of UL148 that prevent cell surface presentation of CD58 but fail to reorganize the ER (67). Likewise, the chimpanzee CMV homolog of UL148 appears to prevent cell surface presentation of CD58 without reorganizing the ER (67).…”

“…Nonetheless, recent work from our laboratory identified charged-cluster-to-alanine mutants of UL148 that prevent cell surface presentation of CD58 but fail to reorganize the ER (67). Likewise, the chimpanzee CMV homolog of UL148 appears to prevent cell surface presentation of CD58 without reorganizing the ER (67). Although we cannot exclude the possibility that ER reorganization may prove crucial for a hitherto unidentified immune evasion function of UL148, these observations argue that ER remodeling is not required for CD58 retention, per se .…”

“…This suggests that UL148 plays essential roles during natural infection. Given that ER reorganization appears to be dispensable for CD58 retention (67), it would not be unreasonable to hypothesize that the striking effects of UL148 on the organelle may reflect additional roles of UL148 that contribute to viral persistence in vivo .…”

Herpesviruses and the Unfolded Protein Response

“…Interestingly, this ER remodeling is enhanced by PERK activation as treatment with ISRIB or a PERK inhibitor delayed the formation of UL148 foci [169]. This remodeling of the ER also appears to be an evolutionary distinct mechanism of human UL140 to control ER reorganization and UPR activation, as the chimpanzee of macaque UL148 do not perform this function [171].…”