Recombinant Human herpesvirus 6B Glycoprotein U20 (U20)

What is HHV-6B U20 and what is its role in viral pathogenesis?

HHV-6B U20 is a glycoprotein encoded by Human Herpesvirus 6B, which infects over 90% of the world's population by age 6 . U20 functions as an immunoevasin, helping the virus evade host immune responses to facilitate lifelong latency. The protein contains an immunoglobulin domain and possesses a class I MHC-like fold .

The primary role of U20 appears to be modulating natural killer (NK) cell activity by binding directly to ULBP1, an NK cell stress ligand recognized by the NKG2D receptor . By masking ULBP1 from recognition, U20 prevents NK cell activation and subsequent killing of infected cells. This mechanism is particularly important because HHV-6B also downregulates class I MHC molecules (MHC-I), which would normally trigger NK cell activation through the "missing-self" recognition pathway .

How does U20 structurally compare to other viral immune evasion proteins?

U20 shares structural similarities with other viral immune evasion proteins, particularly those containing MHC-like folds. Structural modeling of the U20-ULBP1 complex indicates similarities to the m152-RAE1γ complex , a mouse cytomegalovirus immunoevasin that targets NKG2D ligands.

The protein contains an immunoglobulin domain, which is a common feature among immune-regulating molecules . As a type I membrane glycoprotein with a class I MHC-like fold, U20 structurally resembles MHC molecules, which may facilitate its interaction with other immune-related proteins .

What is known about the expression pattern of U20 during HHV-6B infection?

U20 is expressed during active HHV-6B infection with early kinetics, appearing in infected cells as a polypeptide of approximately 100 kDa . Immunoblot analysis of U20 from lysates of HHV-6B-infected SupT1 cells using a polyclonal antibody directed against the cytoplasmic tail of U20 shows this distinct band that is absent in non-infected control cells .

The expression pattern of U20 is consistent with its proposed role as a host defense evasin, as it is located within a block of other host-evasive genes . This coordinated expression with other immune evasion genes suggests a concerted strategy by HHV-6B to modulate host immune responses during infection.

How does the trafficking and posttranslational modification of U20 relate to its immune evasion function?

U20 undergoes complex posttranslational modifications as it traffics through the secretory system, which may be critical for its immune evasion function. After synthesis, U20 initially appears as an 85 kDa polypeptide that is progressively modified to approximately 100 kDa as it moves through the secretory pathway .

The protein receives several key modifications:

• N-linked glycosylation: U20 contains nine N-linked glycans that become processed as the protein traffics through the Golgi apparatus .

• Sialylation: After reaching the trans-Golgi network, at least some of the N-linked glycans become sialylated, as demonstrated by neuraminidase sensitivity studies .

• Phosphorylation: U20 is phosphorylated on at least one Ser, Thr, or Tyr residue .

What is the molecular mechanism by which U20 interferes with NKG2D-ULBP1 interaction?

U20 binds directly to ULBP1 with sub-micromolar affinity, physically masking it from recognition by the NKG2D receptor . This mechanism represents a distinct strategy from other viral immune evasion tactics:

• Unlike some viral proteins that downregulate surface expression of their targets, U20 does not decrease ULBP1 protein levels either at the cell surface or in total cellular content .

• Instead, U20 binding to ULBP1 appears to sterically prevent NKG2D from engaging with ULBP1, as demonstrated by decreased NKG2D binding to ULBP1 at the cell surface in the presence of U20 .

This masking mechanism is observed under multiple experimental conditions:

• In cells transduced with U20

• During actual HHV-6B infection

• When soluble U20 is added to the system

Structural modeling of the U20-ULBP1 complex suggests that U20 may bind to ULBP1 in a manner similar to how mouse cytomegalovirus protein m152 binds to RAE1γ, another NKG2D ligand . This suggests a potential evolutionary conservation of this immune evasion strategy across different herpesvirus species.

How do the functions of HHV-6A U20 and HHV-6B U20 differ despite their 92% sequence identity?

Despite sharing 92% sequence identity, HHV-6A U20 and HHV-6B U20 have been reported to have distinct functions :

• HHV-6A U20 has been shown to downregulate NKG2D ligands, consistent with interfering with NK cell recognition .

• HHV-6B U20 has been reported to inhibit tumor necrosis factor alpha (TNF-α)-induced apoptosis during nonproductive infection with HHV-6B .

• Recent research now shows that HHV-6B U20 also binds to ULBP1, an NKG2D ligand .

This functional divergence despite high sequence similarity raises intriguing questions about the structural determinants of these proteins' functions. The 8% sequence difference may create critical alterations in protein folding, ligand binding sites, or interaction with cellular machinery. Alternatively, the previously reported differences might be context-dependent, with both proteins capable of multiple functions depending on the cellular environment.

Resolving this apparent discrepancy requires further comparative studies of both proteins under identical experimental conditions to determine whether the observed functional differences represent truly distinct mechanisms or context-dependent manifestations of similar underlying activities.

What are the optimal expression systems for producing recombinant HHV-6B U20?

Based on the research data, several expression systems have been successfully used to study U20:

• Lentiviral vectors derived from pHAGE vectors: These have been used to establish stable cell lines expressing untagged or tagged U20 . This system allows for long-term expression studies and cellular trafficking analysis.

• PMA-stimulated expression systems: For SupT1 cell lines with CMV promoter-driven U20 expression, phorbol myristate acetate (PMA) stimulation enhances expression to physiological levels comparable to those seen in HHV-6B-infected cells .

• 293T cell expression: This system allows for robust expression of U20 without requiring PMA stimulation, making it suitable for biochemical characterization .

For functional studies, researchers should consider several tagging strategies:

• Untagged U20 (with I2V substitution to create an ideal Kozak consensus sequence)

• N-terminally TAP-tagged U20 (using a cleavable signal sequence from mouse MHC class I molecule H-2Kb)

• C-terminally TAP-tagged U20

The research indicates that both N- and C-terminally tagged versions of U20 traffic similarly to untagged U20 molecules, suggesting that these tagging strategies do not significantly disrupt protein function .

What methods are effective for detecting U20-ULBP1 interactions and measuring binding affinity?

Several complementary approaches have been used to study U20-ULBP1 interactions:

• Direct binding assays with recombinant proteins: These demonstrate that U20 binds directly to ULBP1 with sub-micromolar affinity .

• Cell-based systems:

  • U20 transduction into target cells

  • HHV-6B infection models

  • Addition of soluble U20 to cell cultures

• Flow cytometry: Using antibodies against the extracellular domain of U20 or tagged versions (TAP-tagged U20) to detect surface expression on non-permeabilized cells .

• NKG2D binding assays: Measuring decreased NKG2D binding to ULBP1 at the cell surface in the presence of U20 provides evidence of the masking effect .

• NK cell activation assays: Demonstrating that U20 expression blocks NK cell activation in response to cell-surface ULBP1 .

• Structural modeling and small-angle X-ray scattering: These techniques have been used to align molecular models of the U20-ULBP1 complex derived from machine-learning approaches .

How can researchers investigate the trafficking pathway of U20 and its posttranslational modifications?

Based on the provided research, several methodological approaches have proven effective for studying U20 trafficking and modifications:

• Pulse-chase analysis: This technique effectively tracks the maturation of U20 from an 85-kDa form to a 100-kDa form as it moves through the secretory pathway .

• Glycosidase treatments:

  • PNGase F treatment to identify N-linked glycans

  • Neuraminidase treatment to detect sialylation of glycans

• Immunofluorescence microscopy: Used to determine the subcellular localization of U20 in both infected cells and those expressing recombinant U20 .

• Flow cytometry of non-permeabilized cells: This method detects surface-expressed U20 using either antibodies against the extracellular domain or tagged versions of the protein .

• Internalization assays: These can measure the rate at which surface-expressed U20 is internalized, as U20 appears to reach the cell surface before being efficiently internalized .

When investigating trafficking, researchers should be aware that:

• U20 trafficking is relatively slow through the secretory system

• The majority of U20 localizes to punctate compartments distinct from lysosomes at steady state

• Only a small portion of U20 is present on the cell surface at any given time due to efficient internalization

What are the critical unexplored aspects of U20 function in HHV-6B pathogenesis?

Several important aspects of U20 function remain to be fully elucidated:

• Broader immune evasion roles: While U20's interaction with ULBP1 is now established, its potential interactions with other immune-related molecules remain largely unexplored .

• Phosphorylation significance: The functional implications of U20 phosphorylation have not been determined. This modification could affect protein-protein interactions, trafficking, or stability .

• Internalization mechanisms: The pathways and molecular machinery involved in U20 internalization from the cell surface, as well as the fate of internalized U20, require further investigation .

• In vivo relevance: The contribution of U20-mediated immune evasion to HHV-6B persistence and pathogenesis in vivo remains to be established through appropriate model systems.

How might structural information about U20 inform therapeutic approaches targeting HHV-6B?

Structural insights into U20 could inform therapeutic development in several ways:

• Structure-based drug design: Detailed structural information about the U20-ULBP1 binding interface could enable the design of small molecules that disrupt this interaction, potentially restoring NK cell surveillance of infected cells .

• Immunotherapeutic approaches: Understanding the structural basis of U20's immune evasion capabilities could inform the development of antibodies or other biologics that neutralize its function without interfering with normal immune interactions.

• Vaccine design: Structural information might identify conserved epitopes in U20 that could serve as targets for vaccine-induced immunity, potentially generating antibodies that block its immune evasion functions.