Immune evasion of human cytomegalovirus : studies of UL18 and US2 function

The β-herpes virus cytomegalovirus (HCMV) infects human populations at a high frequency worldwide. Primary infection with HCMV, usually asymptomatic, is followed by lifelong latency. During the long co-evolution of virus and host, a fine balance has developed between viral immune evasion strategies and defence mechanisms of the immune system. Yet for individuals with a defective, immature, or compromised immune system, HCMV becomes a serious threat. The general aim of this thesis was to investigate functional and molecular aspects of the HCMV-derived immune-evasion proteins UL18 and US2.

One sophisticated immune evasion strategy of HCMV is interference with MHC class I presentation of viral peptides, using several unique short (US) proteins. We dissected the mechanisms underlying the allele specificity of US2 and demonstrated that a single arginine residue at position 181 (Arg181) was critical for US2-mediated inhibition of HLA-A2 cell surface expression. Binding of US2 to HLA-A2 resulted in a unique, large conformational change of the side chain of Arg181. Even though a prerequisite for the interaction of US2 with HLA-A2, the presence of this residue is not sufficient to guarantee binding to other MHC class I alleles.

Another suggested immune modulator with ambiguous function is the protein UL18, a viral MHC class I homologue that associates with β2 microglobulin (β2m). UL18 binds with high affinity to the leukocyte immunoglobulin-like receptor-1 (LIR-1). We tested the impact of several substitutions in UL18 proteins for binding affinity to LIR-1. Our results revealed that residues localized both in the α1 and α3 domain are important for LIR-1 binding, and demonstrated β2m dependency of the UL18/LIR-1 interaction. Finally, two disulfide bridges, one of them unique for UL18, were essential for complex formation of UL18 with β2m.

Since LIR-1 is widely expressed on immune cells, we investigated if UL18 could affect dendritic cells (DCs). We demonstrate that UL18 proteins specifically up- regulated CD83, while not influencing other maturation markers. UL18 also induced IL-10 production and to some extent other cytokines such as TNFα and IL-12. The presence of UL18 during DC maturation via CD40L inhibited DC migration and impaired subsequent T cell responses. We concluded that UL18 can alter phenotype and function of monocyte-derived DC.

The expression of LIR-1 particularly on HCMV specific T cells as well as on NK and T cells in lung-transplanted patients prior to development of CMV caused pneumonia supports the hypothesis that the UL18-LIR-1 interaction may be relevant during natural infection. Therefore, we focused on the role of LIR-1 in the immune response to HCMV and potential effects of UL18 on NK and T cells. Cells infected with a virus lacking the gene for UL18 induced less cytokine responses compared to parental virus, proposing an activating function for UL18. In contrast, isolated UL18 proteins inhibited LIR-1+ T cells, which unlikely played a role in response to infected cells.

In summary, this thesis provides further insights into the mechanism underlying the binding of US2 to HLA-A2 and demonstrates that UL18 interacts with LIR-1 in a manner different from MHC class I ligands. Furthermore, our results regarding the effect of UL18 on several immune cells contributes to a better understanding of the complexity of viral evasion mechanisms.

List of scientific papers

I. Thilo C, Berglund P, Applequist SE, Yewdell JW, Ljunggren HG, Achour A. (2006). "Dissection of the interaction of the human cytomegalovirus-derived US2 protein with major histocompatibility complex class I molecules: prominent role of a single arginine residue in human leukocyte antigen-A2." J Biol Chem 281(13): 8950-7
https://pubmed.ncbi.nlm.nih.gov/16452487

II. Wagner CS, Rölle A, Applequist SE, Cosman D, Ljunggren HG, Berndt K, Achour A. (1970). "Structural Elements Underlying the High Binding Affinity of Human Cytomegalovirus UL18 to Leukocyte Immunoglobulin-like Receptor 1." (Submitted)

III. Wagner CS, Walther-Jallow L, Buentke E, Ljunggren HG, Achour A, Chambers B. (1970). "The Human Cytomegalovirus Protein UL18 Alters the Phenotype and Function of Monocyte-Derived Dendritic Cells." (Submitted)

IV. Wagner CS, Riise GC, Bergstrom T, Karre K, Carbone E, Berg L. (2007). "Increased expression of leukocyte Ig-like receptor-1 and activating role of UL18 in the response to cytomegalovirus infection." J Immunol 178(6): 3536-43
https://pubmed.ncbi.nlm.nih.gov/17339449