Recombinant Human cytomegalovirus Glycoprotein UL18 (H301)

What is Human Cytomegalovirus UL18?

UL18 is a viral homolog of MHC-Ia that is exclusively found in human and great ape cytomegaloviruses but absent in non-human primate CMVs. It functions as an immune evasion protein that binds with high affinity to the inhibitory leukocyte immunoglobulin-like receptor-1 (LIR-1), also known as ILT2, LILRB1, or CD85j. UL18 is extensively glycosylated, with observed molecular weights of approximately 72 and 95 kDa, significantly larger than its predicted weight of 45 kDa due to post-translational modifications .

How does UL18 function in HCMV immune evasion?

UL18 primarily functions by preventing the induction of unconventionally restricted CD8+ T cell responses, specifically those restricted by MHC-E and MHC-II. When UL18 is expressed, it reprograms the CD8+ T cell response from MHC-II and MHC-E restriction to MHC-Ia restriction. This interaction depends on UL18's binding to LIR-1, as demonstrated by experiments with LIR-1-binding mutants that fail to inhibit unconventional T cell priming. This represents a previously unrecognized mechanism of T cell evasion that complements other viral immune evasion strategies .

What experimental systems are used to study UL18 function?

Researchers typically use recombinant cytomegalovirus systems where UL18 can be inserted, modified, or deleted. The key experimental model described in the literature involves inserting human UL18 into rhesus cytomegalovirus (RhCMV) vectors that lack the chemokine-like open reading frames normally preventing unconventional T cell priming. This chimeric approach allows for assessment of UL18 function in non-human primates. In vitro assays using infected fibroblasts co-incubated with T cells from vaccinated animals provide additional insights into the mechanisms of UL18-mediated inhibition .

Mutational Analysis Results

Specific mutations in the LIR-1 binding domain of UL18 abolish its ability to prevent unconventional T cell priming. Two key mutations have been characterized:

Both mutants fail to inhibit MHC-E-restricted CD8+ T cell stimulation in vitro and allow for the induction of MHC-II- and MHC-E-restricted CD8+ T cells in vivo. This strongly indicates that the interaction with LIR-1 is the critical mechanism through which UL18 prevents unconventional T cell responses .

Why does UL18 selectively inhibit unconventional but not conventional T cell responses?

UL18 exhibits a striking selectivity in its immunomodulatory effects. While it effectively prevents the induction of MHC-II- and MHC-E-restricted CD8+ T cells, it does not inhibit conventional MHC-Ia-restricted CD8+ T cell responses. Based on experimental evidence, researchers propose two potential mechanisms for this selectivity:

• The precursor cells for MHC-II- and MHC-E-restricted T cells may express LIR-1, while the naïve T cell pool that gives rise to MHC-Ia-restricted T cells may be LIR-1-negative.

• The TCR signaling strength differs between response types—conventional MHC-Ia-restricted T cells may receive stronger positive TCR signals that can override the negative LIR-1 signal, while the lower-affinity interactions with unconventional CD8+ T cells are more easily inhibited by LIR-1 engagement .

The experimental results from RhCMV vectors expressing UL18 in MHC-evasin-deficient contexts support these hypotheses, as these vectors still stimulate canonical MHC-Ia-restricted T cells despite UL18 expression .

What methodological approaches are used to detect and quantify UL18 expression?

Researchers employ multiple complementary techniques to detect and quantify UL18 expression:

• Immunoblotting of surface biotinylated proteins: This technique reveals the glycosylation pattern of UL18, showing characteristic bands at ~72 and ~95 kDa. The addition of C-terminal tags like FLAG enables specific detection of recombinant UL18 .

• RT-PCR: This method confirms mRNA expression of UL18 in infected cells, verifying successful incorporation and transcription of the UL18 gene in recombinant constructs .

• Next-Generation Sequencing (NGS): Used to validate the construction of recombinant vectors, ensuring the precise incorporation of UL18 or modified UL18 sequences into the viral genome .

• Functional assays: Co-incubation of CD8+ T cells with UL18-expressing or UL18-mutant infected cells, followed by cytokine response measurements, provides functional evidence of UL18 expression and activity .

How should recombinant UL18 constructs be designed for functional studies?

When designing recombinant UL18 constructs for functional studies, researchers should consider:

• Vector Selection: Choose appropriate viral vectors based on experimental goals. For in vivo studies of unconventional T cell priming, 68-1 RhCMV vectors lacking chemokine-like ORFs provide an effective platform .

• Modification Strategies:

  • For epitope tagging: C-terminal FLAG tags can be added without disrupting UL18 function

  • For LIR-1 binding studies: Consider point mutations like D202S that specifically disrupt LIR-1 binding

  • For domain analysis: Target specific functional domains based on structural information

• Validation Methods:

  • Confirm construct integrity by NGS

  • Verify expression by RT-PCR and immunoblotting

  • Assess glycosylation patterns to ensure proper post-translational modification

  • Conduct functional assays to confirm biological activity

• Controls: Include appropriate controls such as wild-type UL18, LIR-1 binding mutants, and empty vectors to distinguish UL18-specific effects from vector-related phenomena .

What are the critical considerations for in vitro assays to assess UL18-mediated inhibition of T cell responses?

When designing in vitro assays to assess UL18's inhibitory effects on T cell responses, researchers should consider:

• Cell Selection: Use appropriate target cells (e.g., fibroblasts) that support CMV infection and express relevant restriction elements. For source of T cells, consider using animals immunized with vectors known to elicit unconventionally restricted responses .

• Readout Systems:

  • Cytokine production (ICS for IFN-γ, TNF-α)

  • Proliferation assays

  • Cytotoxicity measurements

• Controls for Restriction:

  • Include peptide blockers (e.g., VL9 for MHC-E)

  • Use cells with different MHC expressions

  • Compare responses to vectors with/without MHC-E ligands (e.g., Rh67-intact vs. Rh67-deleted)

• Time Course Considerations: Monitor responses at different time points post-infection to capture the dynamic nature of T cell responses .

How might UL18 modifications impact CMV-based vaccine vector development?

The discovery that UL18 prevents unconventionally restricted T cell priming has significant implications for CMV-based vaccine development, particularly for HIV vaccines. Based on research findings, the following considerations are critical:

• Required Modifications: To achieve unconventionally restricted T cell responses in humans using HCMV-based vaccines, the UL18/LIR-1 interaction must be disrupted. This can be accomplished either by:

  • Complete deletion of UL18

  • Introduction of mutations in the LIR-1-binding domain (e.g., D202S)

• Combined Deletions: For optimal unconventional T cell priming, UL18 modifications should be combined with deletions of other inhibitory elements:

  • UL128, UL130 (pentameric complex components)

  • UL146, UL147 (chemokine-like ORFs)

• Immunological Consequences: Properly modified HCMV vectors may elicit MHC-E-restricted CD8+ T cells that could provide broader and more effective control of pathogens like HIV, similar to what has been observed with modified RhCMV vectors against SIV .

What contradictions exist in the current understanding of UL18 function?

Several apparent contradictions in UL18 function require careful consideration:

• Species-Specific Effects: While UL18 effectively inhibits unconventional T cell priming in rhesus macaque models, it remains uncertain whether the same mechanism operates identically in humans. Although rhesus LIR-1 shares approximately 80% identity with human LIR-1 and has similar structural features (four extracellular Ig domains and four intracellular immunoreceptor tyrosine-based inhibitory motifs), subtle differences in binding or signaling might exist .

• Cell Type Disparities: UL18 inhibits unconventional CD8+ T cell priming but does not prevent MHC-Ia-restricted responses. This differential effect is not fully explained by current models and may involve complex interactions between TCR signaling strength and LIR-1 inhibitory signals .

• Evolutionary Questions: It remains unclear why RhCMV does not contain a UL18 homolog if this function is advantageous for viral immune evasion. One hypothesis is that RhCMV uses alternative mechanisms (e.g., chemokine-like ORFs) to achieve the same functional outcome through different molecular interactions .

What methods are recommended for generating UL18 mutants with altered LIR-1 binding?

To generate UL18 mutants with altered LIR-1 binding properties, researchers can employ the following methodological approach:

• Primer-Directed Mutagenesis:

  • Design 50-bp primers carrying sequences homologous to UL18 with the intended modifications

  • Include an additional 50-bp sequence homologous to the upstream or downstream sites of the target sequence

  • Amplify a selection marker (e.g., KanR) and a restriction site (e.g., I-SceI) with these primers

• En Passant Recombination:

  • Introduce the PCR-amplified modified product into a UL18-containing BAC

  • Select for recombinants using the appropriate antibiotic resistance

  • Verify recombination by restriction digest analysis

• Validation:

  • Confirm mutations by Next-Generation Sequencing (NGS)

  • Verify expression by RT-PCR

  • Assess functionality through in vitro binding assays with recombinant LIR-1

This approach has been successfully used to generate UL18-FLAG, UL18-FcRn, and UL18-D202S variants with predictable alterations in LIR-1 binding capability .

What is the recommended workflow for analyzing T cell restriction patterns in response to UL18-expressing vectors?

For analyzing T cell restriction patterns in response to UL18-expressing vectors, researchers should follow this comprehensive workflow:

• Vector Inoculation:

  • Immunize experimental animals with the appropriate vectors (UL18-wild type, UL18-mutant, control vectors)

  • Allow for development of T cell responses (typically 2-4 weeks)

• T Cell Response Characterization:

  • Isolate CD8+ T cells from immunized animals

  • Perform intracellular cytokine staining (ICS) using overlapping peptide pools to quantify total response magnitude

• Restriction Determination:

  • Test for MHC-E restriction using:

    • MHC-E supertope peptides

    • MHC-E blocking with VL9 peptide

    • Stimulation with Rh67-intact vs. Rh67-deleted virus-infected cells

  • Test for MHC-II restriction using:

    • MHC-II supertope peptides

    • MHC-II-blocking antibodies

    • MHC-II-deficient stimulator cells

  • Test for MHC-Ia restriction using:

    • MHC-Ia-blocking antibodies

    • Matched vs. mismatched MHC-Ia cells

    • Known MHC-Ia-restricted epitopes

• Comparative Analysis:

  • Compare restriction patterns between different vector constructs

  • Quantify the proportion of response restricted by each MHC class

  • Analyze response breadth, magnitude, and functionality

This systematic approach enables precise determination of how UL18 and its modified variants affect the restriction pattern of CD8+ T cell responses elicited by viral vectors.