Recombinant Human cytomegalovirus Immediate early glycoprotein (UL37)

What is the basic structure of HCMV UL37 glycoprotein?

HCMV UL37 immediate-early gene encodes a type I membrane-bound glycoprotein (gpUL37) that functions as an integral membrane protein. As a type I membrane protein, gpUL37 has its N-terminus oriented toward the lumen of the endoplasmic reticulum and its C-terminus in the cytoplasm . The protein contains several functional domains, including a leucine zipper motif (aa203-224) and nuclear export signal (aa263-272) in the amino terminus .

The mature form of gpUL37 in HCMV-infected cells migrates as an 83-85 kDa protein on electrophoretic gels, while the unglycosylated form migrates at approximately 68 kDa . The protein undergoes significant post-translational modifications, particularly N-glycosylation, which affects its trafficking and function. Critical functional elements include a region in domain III of the amino terminus (aa296-431) that mediates retrograde axonal transport, and a conserved tyrosine residue at position 480 that facilitates interaction with membrane protein gK .

How is UL37 glycoprotein processed in infected cells?

UL37 glycoprotein undergoes complex processing in HCMV-infected cells. After translation, the protein enters the secretory pathway where it receives N-linked glycosylation modifications. The maturation process involves:

• Initial translocation into the endoplasmic reticulum (ER)

• N-glycosylation within the ER lumen

• Further processing in the Golgi apparatus

• Trafficking to its final destinations

The N-glycosylation pattern of gpUL37 is distinctive - it is resistant to digestion with both endoglycosidase F and endoglycosidase H but sensitive to peptide N-glycosidase F digestion . This glycosylation profile indicates complex processing in both the ER and Golgi apparatus. Confocal microscopy studies have directly demonstrated the passage of gpUL37 through both ER and Golgi by showing colocalization with protein disulfide isomerase (an ER resident protein) and Golgi proteins .

Treatment with tunicamycin, which inhibits N-glycosylation, causes gpUL37 to migrate faster in gel electrophoresis (68 kDa vs. 83-85 kDa), confirming the significant contribution of N-glycans to the protein's apparent molecular weight .

What is the trafficking pathway of UL37 in infected cells?

The trafficking of UL37 involves a sophisticated, multi-step process that is crucial for its various functions. The protein follows this general pathway:

• Initial synthesis and insertion into the ER membrane

• Processing through the ER with N-glycosylation

• Transit through mitochondrion-associated membranes (MAMs)

• Final localization to the outer mitochondrial membrane (OMM)

This sequential trafficking is particularly well-documented for the pUL37x1 isoform (vMIA). Confocal microscopy and subcellular fractionation studies have verified that gpUL37 colocalizes with ER markers before progressing to the Golgi network . During this transit, the protein associates with lipid rafts in the ER and MAM compartments . These lipid raft associations are dependent on cholesterol binding capacity, which appears to be a conserved feature of UL37 proteins .

The protein's trafficking to mitochondria is governed by distinct mitochondrial targeting signals that are functionally separable from its lipid raft association mechanisms . Once at the outer mitochondrial membrane, pUL37x1/vMIA exerts its anti-apoptotic functions in certain cell types.

How does UL37 associate with internal lipid rafts, and what is the significance?

UL37, particularly the pUL37x1 isoform, demonstrates a unique association with internal lipid rafts (LRs) within the endoplasmic reticulum and mitochondrion-associated membranes. This association has been experimentally verified through:

• Extraction with methyl-β-cyclodextrin (MβCD), which removes pUL37x1/vMIA from lysed but not intact cells, indicating association with internal rather than plasma membrane LRs

• Isolation of detergent-resistant membranes (DRMs) from purified intracellular organelles, which confirms localization within ER/MAM LRs

• Absence of pUL37x1/vMIA in DRMs from mitochondria, suggesting the LR association is limited to specific compartments

The significance of this lipid raft association lies in its persistence throughout all temporal phases of HCMV infection, suggesting its importance for viral growth . This association depends on cholesterol binding domains rather than mitochondrial targeting signals. Specifically, mutation studies have identified cholesterol binding domains in the UL37x1 leader sequence that are critical for this interaction .

The lipid raft association may facilitate:

• Concentration of viral proteins in specific membrane microdomains

• Organization of protein complexes required for viral assembly

• Modulation of host signaling pathways from these specialized membrane domains

What are the essential functions of UL37 in HCMV replication?

UL37 serves multiple essential functions during HCMV infection that vary by cell type:

In human fibroblasts (HFFs), pUL37x1/vMIA functions as a potent anti-apoptotic protein that protects infected cells from both apoptosis and caspase-independent mitochondrial serine protease-mediated cell death . This protection extends the viability of infected cells, allowing for maximal viral replication. Mutations in UL37x1 result in growth defects in HFFs, highlighting its importance in this cell type .

Interestingly, in human neural precursor cells (hNPCs), pUL37x1/vMIA does not protect against HCMV-induced cell death, despite trafficking efficiently to mitochondria . Even more surprisingly, UL37x1 mutants produce infectious progeny in hNPCs with similar kinetics and levels as wild-type virus, suggesting that pUL37x1/vMIA is not essential for HCMV growth in this cell type .

Research with pseudorabies virus (PrV), another herpesvirus, provides additional insight into UL37 function. In PrV, UL37 is critical for:

• Secondary envelopment in the cytoplasm

• Proper tegumentation and viral assembly

• Efficient production of infectious particles

Without UL37, PrV exhibits impaired replication with final titers approximately 100-fold lower than wild-type virus .

How does UL37 contribute to mitochondrial function and apoptosis inhibition?

The pUL37x1 isoform (vMIA) has a well-established role in regulating mitochondrial function and inhibiting apoptosis, though this activity shows cell-type specificity:

Mechanism of action:

• pUL37x1/vMIA traffics sequentially from the ER through MAMs to the outer mitochondrial membrane

• At the mitochondria, it inhibits cytochrome c release, preventing activation of the intrinsic apoptotic pathway

• This protection extends to both caspase-dependent apoptosis and caspase-independent mitochondrial cell death pathways

Quantitative analysis shows that in hNPCs, both wild-type HCMV and a UL37x1 mutant (BADsubUL37x1) induce similar levels of cell death:

• At 5 days post-infection: 15.3% Annexin V+ cells in wild-type infection vs. 19.6% in UL37x1 mutant infection

• At 8 days post-infection: 58.5% Annexin V+ cells in wild-type infection vs. 43.6% in UL37x1 mutant infection

These findings suggest that pUL37x1/vMIA function is influenced by cellular context, potentially due to different mitochondrial compositions or regulatory pathways in various cell types.

What are the optimal methods for producing recombinant UL37 glycoprotein for research?

When producing recombinant UL37 glycoprotein for research purposes, several methodological approaches can be employed, each with specific advantages:

Expression systems:

• Mammalian cell expression: Preferred for obtaining naturally glycosylated and properly folded UL37. HEK293 or CHO cells are commonly used for this purpose. These systems allow for proper post-translational modifications, particularly the complex N-glycosylation observed in HCMV-infected cells .

• Bacterial expression of fragments: For structural studies of specific domains, bacterial expression of UL37 fragments fused to tags like GST can be useful. This approach was successfully employed for creating a monospecific polyclonal rabbit antiserum against a bacterial glutathione S-transferase (GST)-UL36 fusion protein .

Purification strategy:

• Initial isolation via affinity chromatography (His-tag or specific antibody-based)

• Further purification using ion exchange chromatography

• Final polishing with size exclusion chromatography to obtain homogeneous protein

Critical considerations:

• Due to UL37's membrane association, detergent solubilization is typically required

• For optimal activity, the choice of detergent is critical - milder detergents like DDM or CHAPS are preferable for maintaining structure

• When studying lipid raft associations, special care must be taken to preserve these interactions during purification

Verification methods:

• Western blotting with UL37-specific antibodies

• Glycosylation analysis using endoglycosidase and PNGase F treatment

• Mass spectrometry to confirm protein identity and modifications

How can researchers effectively study UL37 trafficking in living cells?

Studying UL37 trafficking in living cells requires sophisticated approaches to visualize and track this dynamic protein. Several effective methodologies include:

Fluorescent protein tagging:

• GFP or mCherry fusion constructs of UL37 for live-cell imaging

• Care must be taken to ensure tags don't interfere with trafficking signals

• C-terminal tagging is often preferable since the N-terminus contains ER translocation signals

Organelle co-localization studies:
As demonstrated in the literature, co-localization with organelle markers provides valuable insights:

• Protein disulfide isomerase as an ER marker

• Golgi-specific proteins for Golgi localization

• Mitotracker or mitochondrial-targeted fluorescent proteins for mitochondrial tracking

Advanced microscopy techniques:

• Confocal microscopy for high-resolution localization studies

• Live-cell time-lapse imaging to track protein movement in real-time

• Super-resolution microscopy (STORM, PALM, or STED) for detailed subdomain localization

• Fluorescence recovery after photobleaching (FRAP) to assess protein mobility within membranes

Biochemical approaches:

• Subcellular fractionation to isolate specific organelles (ER, MAM, mitochondria)

• Analysis of detergent-resistant membrane fractions to study lipid raft association

• Density gradient centrifugation to separate different membrane compartments

Inhibitor treatments:

• Brefeldin A to disrupt ER-Golgi trafficking

• Nocodazole to disrupt microtubule-dependent transport

• Methyl-β-cyclodextrin to deplete cholesterol and disrupt lipid rafts

• Tunicamycin to inhibit N-glycosylation and assess its impact on trafficking

How do specific domains of UL37 contribute to its diverse functions?

UL37 contains multiple functional domains that contribute to its diverse activities in viral replication, trafficking, and host defense modulation:

N-terminal domains:

• Leucine zipper motif (aa203-224): Likely mediates protein-protein interactions critical for UL37 function

• Nuclear export signal (aa263-272): Controls protein localization between nuclear and cytoplasmic compartments

• R2 region in domain III (aa296-431): Critical for retrograde axonal transport within neurons, particularly important for neurotropic herpesviruses

Central region:

• Tyrosine 480 (Y480): Plays a key role in interaction with membrane protein gK, which is essential for cytoplasmic virion envelopment and viral replication

C-terminal domains:

• Dystonin interaction domain (aa578-899): Mediates binding to dystonin (BPAG1), a non-motor protein that bridges cytoskeleton networks for both retrograde and anterograde transport in various cell types

• Deamidation pseudoenzyme domain: Contains two critical cysteine residues (C819 and C850) that facilitate post-translational modification called deamidation on pathogen recognition receptors cGAS and RIG-I, affecting innate immune responses

• TRAF6-binding domain (aa1099-1104): Located at the tail end of the C-terminus, this domain activates NFκB signaling, potentially modulating host immune responses

Cholesterol binding domains:

• Located in the UL37x1 leader sequence, these domains are critical for lipid raft association but functionally distinct from mitochondrial targeting signals

The multi-domain structure of UL37 explains its versatility in performing diverse functions throughout the viral life cycle, from capsid transport to immune evasion.

What structural features govern UL37's interaction with viral and cellular proteins?

UL37 engages in multiple protein-protein interactions that are critical for its various functions. These interactions are governed by specific structural features:

Viral protein interactions:

• UL36 interaction: UL37 associates with the major tegument protein UL36, forming a complex essential for virus maturation, specifically for outer tegumentation and secondary envelopment . This interaction likely involves multiple contact points and conformational complementarity.

• gK interaction: The tyrosine residue at position 480 (Y480) mediates interaction with the viral membrane protein gK, which is critical for cytoplasmic virion envelopment .

• VP5 interaction: UL37 interacts with VP5 (major capsid protein) and together they bind to dynein intermediate chain (DIC), facilitating capsid transport to the nucleus .

• Tegument assembly: In the absence of UL37, other tegument proteins like UL49 fail to properly associate with capsids, indicating that UL37 provides a foundation for sequential tegument assembly .

Cellular protein interactions:

• Dystonin (BPAG1) binding: Amino acids 578-899 in the C-terminus mediate interaction with dystonin, a cytoskeletal crosslinking protein involved in retrograde and anterograde transport in various cell types .

• Dynein interaction: Together with VP5, UL37 interacts with dynein intermediate chain to facilitate capsid transport along microtubules .

• Deamidation of innate immune sensors: UL37 functions as a pseudoenzyme that can deamidate the pathogen recognition receptors cGAS and RIG-I through its two critical cysteine residues (C819 and C850) .

• TRAF6 binding: The C-terminal TRAF6-binding domain allows UL37 to manipulate NFκB signaling, potentially modulating inflammatory responses .

These structural interactions highlight UL37's role as a central organizing protein that coordinates viral assembly and simultaneously manipulates host cell mechanisms for optimal viral replication.

How does HCMV UL37 differ from homologous proteins in other herpesviruses?

HCMV UL37 shares core structural and functional features with homologous proteins in other herpesviruses, but also exhibits significant differences:

Structural similarities:

• All herpesvirus UL37 homologs function as tegument proteins

• Most contain domains for capsid binding and intracellular transport

• Many exhibit interactions with the major tegument protein UL36

Functional similarities:

• Role in secondary envelopment and virion assembly

• Involvement in intracellular capsid transport

• Contribution to viral egress

Key differences:

• Size and complexity: HCMV UL37 is generally larger and more complex than its alphaherpesvirus counterparts.

• Anti-apoptotic function: HCMV UL37x1 (vMIA) has a well-documented anti-apoptotic function through mitochondrial targeting, which is not as prominent in other herpesvirus UL37 homologs .

• Cell-type specificity: HCMV UL37x1 shows cell-type specificity, functioning as an anti-apoptotic protein in fibroblasts but not in neural precursor cells . This level of cell-type dependent function variation has not been as thoroughly documented in other herpesviruses.

• Lipid raft association: HCMV UL37 associates with internal lipid rafts in the ER/MAM, a property that has been specifically documented for this virus .

• Immune modulation: HCMV UL37 contains specific domains for deamidation of innate immune sensors and NFκB modulation that may not be conserved across all herpesvirus UL37 proteins .

Pseudorabies virus (PrV) UL37 provides an interesting comparison: while it shares the essential role in secondary envelopment and virion assembly, PrV lacking UL37 can still produce L-particles (enveloped particles lacking capsids), suggesting some distinct mechanistic differences in how different herpesvirus UL37 proteins function in virion assembly .

What experimental approaches best reveal the essential versus cell-type specific functions of UL37?

Understanding the essential versus cell-type specific functions of UL37 requires sophisticated experimental approaches that can dissect its multifunctional nature:

Genetic approaches:

• Targeted mutagenesis: Creating point mutations or deletions in specific functional domains helps identify which regions are universally required versus those needed only in certain contexts . For example, mutations in cholesterol binding domains demonstrated their role in lipid raft association .

• Complementation assays: Testing whether UL37 mutants can be rescued by wild-type protein expression helps identify truly essential functions .

• Cell-type comparative analysis: Studying the same UL37 mutants in different cell types reveals context-dependent functions. The BADsubUL37x1 mutant showed dramatic growth defects in HFFs but replicated efficiently in hNPCs, revealing unexpected cell-type specificity .

Biochemical approaches:

• Interaction proteomics: Mass spectrometry analysis of UL37 binding partners in different cell types can reveal cell-specific interaction networks.

• Glycosylation analysis: Comparison of UL37 glycosylation patterns across cell types using endoglycosidase treatments can reveal differences in protein processing .

• Subcellular fractionation: Isolating different cellular compartments (ER, MAM, mitochondria) to track UL37 distribution in various cell types .

Functional assays:

• Viral growth curves: Comparing replication kinetics of wild-type and mutant viruses across cell types:

  • In hNPCs (MOI=1), BADsubUL37x1 produced similar viral titers to wild-type HCMV (approximately 10^5 PFU/well)

  • At lower MOI (0.3), BADsubUL37x1 showed delayed growth in hNPCs but eventually reached similar levels to revertant virus (1.5×10^5 PFU)

  • In contrast, the same mutant is severely growth-impaired in HFFs

• Apoptosis assays: Using Annexin V staining to quantify cell death:

  • In hNPCs, similar levels of apoptosis were observed between wild-type HCMV (58.5% Annexin V+ cells at 8 days post-infection) and UL37x1 mutant (43.6%)

• Microscopy-based trafficking studies: Comparing UL37 localization patterns and dynamics across cell types using confocal microscopy and co-localization with organelle markers .

These complementary approaches collectively provide a comprehensive understanding of which UL37 functions are universally essential versus those that display cell-type specificity.

How might targeting UL37 glycoprotein inform novel antiviral strategies?

UL37 glycoprotein presents several promising avenues for antiviral development due to its essential roles in multiple aspects of the viral life cycle:

Potential targeting approaches:

• Inhibition of UL37 trafficking: Compounds that disrupt UL37's sequential trafficking from ER through MAMs to mitochondria could prevent its functional localization . Small molecules that interfere with specific sorting signals or transport machinery could be effective without directly targeting the protein.

• Disruption of cholesterol binding: Since UL37's lipid raft association depends on cholesterol binding and occurs throughout all temporal phases of HCMV infection, compounds that specifically block this interaction could disrupt multiple viral functions . Unlike general cholesterol-depleting agents, targeted inhibitors of the cholesterol binding domains would have more specific antiviral effects.

• Blocking key protein-protein interactions: Inhibitors that prevent UL37's interaction with:

  • UL36, disrupting tegument assembly

  • gK (via Y480), interfering with virion envelopment

  • Dystonin, impairing intracellular transport

  • TRAF6, preventing manipulation of host NFκB signaling

• Cell-type specific approaches: The differential requirement for UL37 functions in various cell types suggests potential for targeted therapies . For instance, antivirals targeting UL37's anti-apoptotic function might be most effective in fibroblasts but less so in neural cells.

Advantages of UL37 as a target:

• Essential for viral replication in many cell types

• Involved in multiple stages of viral life cycle

• Contains conserved domains that could be targeted with broad-spectrum antivirals

• Has both structural and enzymatic functions that can be inhibited in different ways

Considerations for antiviral development:

• Need to consider cell-type specific effects, as demonstrated by differential requirements in fibroblasts versus neural cells

• Potential for viral escape mutations must be evaluated

• Compounds must distinguish between viral UL37 and any similar host proteins to avoid toxicity

The multifunctional nature of UL37 makes it particularly attractive as an antiviral target, as disruption of even one of its essential functions could significantly impair viral replication.

What methodological approaches are most effective for screening inhibitors of UL37 function?

Developing effective inhibitors of UL37 function requires robust screening methodologies that can identify compounds disrupting its various activities:

High-throughput primary screens:

• Protein-protein interaction assays:

  • FRET or BRET-based assays to monitor interactions between UL37 and key binding partners like UL36 or gK

  • Split-luciferase complementation assays for detecting disruption of specific protein interactions

  • AlphaScreen technology to identify compounds that block defined interaction interfaces

• Trafficking disruption screens:

  • High-content imaging using fluorescently tagged UL37 to monitor proper localization

  • Automated microscopy platforms to quantify colocalization with organelle markers (ER, Golgi, mitochondria)

  • FACS-based sorting of cells showing altered UL37 localization patterns

• Lipid raft association screens:

  • Assays measuring UL37 partitioning into detergent-resistant membrane fractions

  • Fluorescence-based techniques monitoring cholesterol binding to UL37 domains

  • Displacement assays using labeled cholesterol analogs

Secondary validation assays:

• Viral replication assays:

  • Multi-day growth curve analysis in relevant cell types

  • Plaque reduction assays to quantify antiviral efficacy

  • Comparative analysis in different cell types (e.g., fibroblasts vs. neural cells) to assess cell-type specific effects

• Mechanistic validation:

  • Western blotting to assess proper processing and glycosylation of UL37

  • Subcellular fractionation to confirm disruption of proper localization

  • Electron microscopy to visualize effects on viral assembly and secondary envelopment

• Resistance profiling:

  • Selection and sequencing of resistant viral variants

  • Mapping of resistance mutations to specific UL37 domains

  • Cross-resistance testing against different UL37-targeting compounds

Advanced screening technologies:

• Fragment-based drug discovery:

  • NMR or X-ray crystallography screening of fragment libraries against purified UL37 domains

  • Structure-based optimization of fragment hits

• DNA-encoded library technology:

  • Screening vast chemical libraries against immobilized UL37 protein

  • Selection of binders through affinity-based methods and DNA barcode sequencing

• Virtual screening:

  • Molecular docking against structural models of UL37 functional domains

  • Molecular dynamics simulations to identify stable binding modes

  • Structure-based design of compounds targeting specific UL37 functions

The most effective screening strategies will combine multiple approaches to identify diverse inhibitors targeting different aspects of UL37 function, followed by rigorous validation in relevant cellular models of HCMV infection.

What are the current technical limitations in studying UL37, and how might they be overcome?

Research on UL37 faces several technical challenges that limit our comprehensive understanding of this multifunctional protein:

Current limitations:

• Structural complexity: As a large membrane glycoprotein with multiple domains, obtaining structural information about full-length UL37 has been challenging .
  Solution: Employ cryo-electron microscopy for membrane proteins or focus on solving structures of discrete functional domains using X-ray crystallography or NMR.

• Glycosylation heterogeneity: The complex N-glycosylation pattern of UL37 creates heterogeneity that complicates structural and functional analyses .
  Solution: Use glycosidase treatments, glycosylation site mutations, or expression in glycosylation-modified cell lines to produce more homogeneous protein for analysis.

• Membrane association: UL37's integral membrane nature makes it difficult to purify in its native state .
  Solution: Develop improved detergent solubilization protocols or nanodiscs/amphipol systems to maintain native membrane environment during purification.

• Multi-compartment trafficking: Tracking UL37's dynamic movement between cellular compartments requires sophisticated imaging approaches .
  Solution: Implement advanced live-cell super-resolution microscopy with minimal photobleaching and optimized fluorescent tagging strategies.

• Cell-type specific functions: The differential behavior of UL37 in various cell types complicates the generalization of research findings .
  Solution: Systematically study UL37 across a diverse panel of relevant cell types, including primary cells derived from tissues important in HCMV pathogenesis.

• Distinguishing direct from indirect effects: As a multifunctional protein, determining which phenotypes are directly attributable to specific UL37 functions is challenging.
  Solution: Create comprehensive libraries of domain-specific mutants and employ precise genome editing to introduce these mutations into the viral genome.

• Authentic in vitro systems: Current in vitro systems may not fully recapitulate the complex environment in which UL37 functions during infection.
  Solution: Develop organoid or tissue-like culture systems that better reflect in vivo complexity, particularly for neural tissues where UL37 exhibits unique properties .

What are the most promising future research directions for UL37 glycoprotein studies?

Several promising research directions could significantly advance our understanding of UL37 glycoprotein and its potential as a therapeutic target:

Emerging research opportunities:

• Structural biology breakthroughs: Applying advances in cryo-EM and integrative structural biology to:

  • Determine the complete structure of UL37 in membrane environments

  • Visualize UL37 in complex with binding partners like UL36, gK, or host proteins

  • Map conformational changes during trafficking and maturation

• Single-molecule studies: Utilizing advanced microscopy to:

  • Track individual UL37 molecules during trafficking between cellular compartments

  • Measure kinetics of protein-protein interactions in living cells

  • Correlate molecular behaviors with functional outcomes

• Systems biology approaches: Employing multi-omics strategies to:

  • Map the complete UL37 interactome across different cell types and infection stages

  • Identify cell-type specific determinants of UL37 function

  • Create predictive models of how UL37 functions within the viral replication network

• Translational applications: Developing UL37-focused interventions:

  • Design peptide inhibitors targeting specific UL37 functional domains

  • Create decoy molecules that mimic UL37 binding partners

  • Develop antibodies or nanobodies that recognize accessible UL37 epitopes

• Host-pathogen interface studies: Investigating how UL37:

  • Modulates mitochondrial function in different cellular contexts

  • Interacts with innate immune signaling components

  • Contributes to cell-type tropism and HCMV pathogenesis

• Comparative virology: Expanding research to:

  • Understand functional conservation and divergence among UL37 homologs

  • Identify common mechanisms that could be targeted by broad-spectrum antivirals

  • Explore how UL37 functions evolved to adapt to specific host cell environments

• Innovative technologies: Applying cutting-edge approaches:

  • CRISPR-based screening to identify host factors required for UL37 function

  • Proximity labeling techniques to map UL37's dynamic protein neighborhood

  • Artificial intelligence to predict functional consequences of UL37 mutations

The multi-functional nature of UL37 positions it at the intersection of viral assembly, trafficking, and host defense modulation. Future research that integrates structural insights with functional analyses across relevant cell types will be particularly valuable for understanding both basic herpesvirus biology and developing targeted interventions.