Recombinant Human cytomegalovirus Uncharacterized protein UL10 (UL10)

What is UL10 and how is it classified within the HCMV genome?

UL10 is a protein encoded by the Human Cytomegalovirus (HCMV) that has been classified as a member of the HCMV RL11 gene family. While pUL10 is dispensable for viral replication in cultured cells, its amino acid sequence is well conserved among different HCMV isolates, suggesting the protein plays a crucial role in viral survival in the host environment. This conservation pattern indicates strong evolutionary pressure to maintain UL10 functionality, likely due to its importance in viral immune evasion strategies .

Methodological approach: To study UL10's genomic classification, researchers should perform comparative genomic analysis across multiple HCMV strains using next-generation sequencing and bioinformatics tools. Sequence alignment and phylogenetic analysis can help determine evolutionary relationships and conservation patterns within the RL11 family.

What are the primary immunomodulatory functions of UL10?

Research demonstrates that UL10 encodes a protein (pUL10) with significant immunosuppressive properties. Studies have shown that pUL10 is cleaved from the cell surface of both fibroblasts and epithelial cells and interacts with a cellular receptor ubiquitously expressed on human leukocytes. When peripheral blood mononuclear cells are preincubated with purified pUL10 ectodomain, they show significantly impaired proliferation and substantially reduced pro-inflammatory cytokine production, particularly in CD4+ T cells upon in vitro stimulation. Additionally, the inhibitory effect of pUL10 is observed on antigen receptor-mediated intracellular tyrosine phosphorylation in T-cell lines .

Methodological approach: To assess UL10's immunomodulatory functions, design experiments using purified recombinant pUL10 protein with lymphocyte proliferation assays, cytokine profiling by ELISA or flow cytometry, and phosphorylation analysis by western blotting.

How does UL10 expression vary across different cell types during infection?

Functional profiling studies of the HCMV genome have revealed that UL10 demonstrates cell type-specific functions. A UL10 deletion mutant grew normally in human foreskin fibroblasts (HFF) and human microvascular endothelial cells (HMVEC) but reached a 500-fold higher titer than wild-type Towne BAC in retinal pigment epithelial (RPE) cells. This observation implies that UL10 encodes cell type-specific functions for virus-growth inhibition, particularly in RPE cells .

Methodological approach: To study cell type-specific expression patterns, perform multi-cell type infection experiments using reporter-tagged UL10 constructs or quantitative RT-PCR combined with immunohistochemistry to visualize expression in different tissues.

What expression systems are optimal for producing recombinant pUL10 for functional studies?

Based on protocols used for other viral proteins, several expression systems can be adapted for recombinant pUL10 production:

Bacterial expression system: While convenient, this system may not provide proper post-translational modifications crucial for pUL10 function. If using this approach, consider fusion with His-tagged SUMO protein to improve solubility, followed by affinity purification using Ni²⁺-chelate chromatography .

Baculovirus expression system: This system offers advantages for producing eukaryotic proteins with proper folding and modifications. The method typically achieves 30-90% purity with approximately 25-30% recovery of activity. Purification strategies include hydrophobic interaction chromatography, L-tyrosine-agarose column chromatography, and blue A resin separation .

Mammalian expression system: For optimal post-translational modifications, particularly glycosylation patterns that may be critical for receptor binding, consider using HEK293 or CHO cells with efficient transfection methods and stable selection.

Table 1: Comparison of Expression Systems for Recombinant pUL10 Production

Methodological approach: For optimal functional studies, express the extracellular domain of pUL10 using a mammalian expression system with appropriate glycosylation capabilities, followed by affinity purification and size exclusion chromatography to ensure biological activity .

How can researchers design experiments to identify pUL10's cellular receptor?

Identifying pUL10's cellular receptor requires a multi-faceted approach:

• Protein-protein interaction screenings: Utilize techniques such as yeast two-hybrid screening, co-immunoprecipitation followed by mass spectrometry, or protein microarrays to identify potential binding partners from human leukocyte lysates.

• Flow cytometry-based binding assays: Develop fluorescently labeled recombinant pUL10 for binding studies with various cell types, followed by competition assays with monoclonal antibodies against candidate receptors to confirm specificity .

• Cross-linking studies: Employ chemical cross-linking of pUL10 to its binding partners on cell surfaces, followed by purification and mass spectrometric analysis to identify the receptor.

• CRISPR-Cas9 screening: Perform genome-wide CRISPR screens in susceptible cell lines to identify genes required for pUL10 binding or functional effects.

Methodological approach: Initial identification of candidate receptors should use recombinant pUL10 ectodomain as bait in pull-down assays with membrane fractions from human leukocytes, followed by mass spectrometry analysis. Confirmation should include both binding assays and functional validation using receptor-knockout cell lines .

What methods can resolve contradictions in reported UL10 functions across different experimental systems?

Research findings have shown contradictory results regarding UL10's function in different cell types and experimental systems. To address these contradictions, researchers should:

• Standardize experimental conditions: Use consistent viral strains, cell types, and infection parameters across studies to minimize variables that could lead to contradictory results.

• Employ Boolean rule-based analysis: Apply structured contradiction pattern analysis as described by Gitomer and Crouse (2019) to identify potential sources of conflicting data. This approach uses parameters (α, β, θ) where α represents the number of interdependent items, β represents the number of contradictory dependencies, and θ represents the minimal number of required Boolean rules to assess these contradictions .

• Multi-cell type comparative studies: Conduct parallel experiments in different cell types (HFF, HMVEC, RPE) under identical conditions to directly compare UL10 functions and effects.

• Temporal analysis: Examine UL10 expression and function at different time points post-infection to determine if contradictions are due to temporal differences in activity.

Methodological approach: Design comprehensive experiments that simultaneously test UL10 function across multiple cell types, time points, and functional readouts. Use statistical approaches like two-way ANOVA to analyze interaction effects between variables that might explain contradictory findings .

How can researchers effectively design experiments to study UL10's interaction with the host immune system?

To study UL10's interaction with the immune system, researchers should consider:

• Ex vivo cell-based assays: Utilize purified recombinant pUL10 in immune cell functional assays measuring:

  • T-cell proliferation using CFSE dilution or 3H-thymidine incorporation

  • Cytokine production profiles using multiplex bead arrays or intracellular cytokine staining

  • Changes in cell surface activation markers by flow cytometry

• Signal transduction analysis: Examine the effects of pUL10 on key signaling pathways:

  • Phosphorylation status of tyrosine kinases (e.g., p56lck) and downstream signaling molecules

  • Transcription factor activation (NFAT, NF-κB)

  • Gene expression changes using RNA-seq or targeted RT-PCR panels

• Receptor blocking studies: Use antibodies against candidate receptors or soluble receptor competitors to block pUL10 effects and confirm specificity of interactions .

Methodological approach: Isolate primary human immune cell subsets (CD4+ T cells, CD8+ T cells, B cells, monocytes), pretreat with purified pUL10 at physiologically relevant concentrations, then stimulate with appropriate activators and measure functional outcomes including proliferation, cytokine production, and signaling pathway activation .

What approaches can determine the structural features of pUL10 critical for its immunosuppressive function?

Understanding the structure-function relationship of pUL10 requires:

• Protein structure determination: Similar to approaches used for the UL111A-encoded viral IL-10 and UL10 homologs, use X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy to determine the three-dimensional structure of pUL10 .

• Mutagenesis studies: Create a panel of pUL10 mutants with targeted substitutions or deletions in conserved domains, then assess each variant's ability to:

  • Bind its cellular receptor(s)

  • Inhibit T-cell proliferation

  • Suppress cytokine production

  • Alter signal transduction pathways

• Domain mapping: Generate truncated versions of pUL10 to identify minimal functional domains required for immunosuppressive activity.

• Glycosylation analysis: Investigate the role of post-translational modifications using lectin affinity chromatography (LAC) with Concanavalin A (Con A) and wheat germ agglutinin (WGA) to enrich different glycoforms, followed by functional testing .

Methodological approach: Express the full-length pUL10 and systematically designed truncation/mutation variants, purify each using affinity chromatography, and test their functional activity in parallel assays measuring immune cell activation and signaling. Correlate structural features with functional outcomes to map critical domains .

How does UL10 contribute to HCMV latency and reactivation?

While direct research on UL10's role in latency is limited, its immunomodulatory functions suggest potential involvement in this process. By comparison with other HCMV immune evasion genes like UL111A (viral IL-10):

• Latency establishment: pUL10 may contribute to establishing latency by suppressing T-cell responses that would otherwise clear infected cells.

• Maintenance mechanisms: Similar to LAcmvIL-10 (a viral IL-10 isoform), UL10 might induce changes in the host cell environment that promote survival of latently infected cells .

• Reactivation triggers: The cell type-specific functions of UL10 may play a role in determining which tissues support viral reactivation.

Methodological approach: Study UL10 expression in experimental latency models using CD34+ hematopoietic progenitor cells or monocytes. Compare wild-type and UL10-deletion mutant viruses for their ability to establish, maintain, and reactivate from latency. Analyze transcriptional and epigenetic changes induced by UL10 expression during these phases .

How can researchers effectively study the cell type-specific functions of UL10?

To investigate why UL10 shows different effects across cell types:

• Comparative proteomics: Identify differences in receptor expression or downstream signaling components across cell types (HFF, HMVEC, RPE) using quantitative proteomics approaches.

• Cell type-specific knockout models: Generate receptor-knockout cell lines for each tissue type to determine if differential receptor expression explains cell type-specific effects.

• Transcriptional profiling: Perform RNA-seq on different cell types with and without UL10 expression to identify cell-specific transcriptional responses.

• In vivo tissue-specific models: Develop animal models with tissue-specific expression or deletion of UL10 homologs to assess in vivo relevance.

Table 2: Observed Effects of UL10 Deletion in Different Cell Types

Methodological approach: Perform parallel infections of multiple cell types with wild-type and UL10-deletion mutant viruses, then use systems biology approaches (transcriptomics, proteomics, metabolomics) to create comprehensive models of cell type-specific responses that could explain the differential effects .

What glycoproteomic approaches can identify post-translational modifications of pUL10?

Glycosylation and other post-translational modifications may be critical for pUL10 function. Researchers should consider:

• Lectin affinity chromatography (LAC): Use lectins with different glycan specificities (Con A for high-mannose N-glycans, WGA for N-acetyl-glucosamine) to enrich glycoforms of pUL10 .

• Mass spectrometry analysis: Apply stable isotope dimethyl labeling combined with 2D liquid chromatography separation for quantitative mass spectrometric analysis of glycopeptides .

• Site-directed mutagenesis: Identify putative glycosylation sites through sequence analysis and create site-specific mutants to assess the functional importance of each site.

• Glycosidase treatments: Use enzymatic deglycosylation (PNGase F, Endo H) to remove specific glycan types followed by functional testing to determine their contribution to activity.

Methodological approach: Express recombinant pUL10 in mammalian cells, purify using a combination of affinity chromatography and LAC, then perform glycoproteomics analysis using tandem mass spectrometry to map the glycosylation sites and glycan structures. Validate findings using site-directed mutagenesis of potential glycosylation sites followed by functional testing .

How can CRISPR-Cas9 technology be applied to study UL10 functions?

CRISPR-Cas9 technology offers powerful approaches for UL10 research:

• Viral genome editing: Generate precise UL10 mutations or deletions in the context of the complete HCMV genome using CRISPR-Cas9 editing of viral BAC clones.

• Host factor screening: Perform genome-wide or targeted CRISPR screens to identify host factors required for UL10 expression, processing, or function.

• Receptor identification: Create knockout cell libraries targeting candidate receptors to identify the cellular binding partner(s) of pUL10.

• Tagged UL10 variants: Introduce epitope tags or fluorescent protein fusions at the endogenous UL10 locus to study protein localization and interactions without overexpression artifacts.

Methodological approach: Design guide RNAs targeting UL10 or host factors of interest, establish appropriate delivery methods for the target cell types, validate editing efficiency, and then perform functional assays to assess the impact of the genetic modifications on viral replication, immune evasion, and host cell responses.