Recombinant Human herpesvirus 2 Envelope protein US9 (US9)

What is the basic structure and cellular location of HSV-2 US9 protein?

HSV-2 US9 is a type II membrane protein encoded by the US9 gene. It has a predicted molecular weight of approximately 10,000 Da, though on denaturing polyacrylamide gels it forms multiple overlapping bands ranging from 12,000 to 25,000 Da. The protein contains an amino-terminal cytoplasmic tail with important trafficking motifs, including an acidic cluster containing putative phosphorylation sites and a dileucine endocytosis signal .

In infected cells, US9 is primarily localized to the trans-Golgi network (TGN) region, but its distribution is dynamic, involving continuous recycling between the TGN and plasma membrane. This trafficking is mediated by endocytosis signals in its cytoplasmic domain. When incorporated into virions, US9 becomes a component of the tegument, the proteinaceous layer between the capsid and envelope .

How is US9 protein conserved across alphaherpesviruses?

The US9 gene is highly conserved among the alpha subfamily of herpesviruses, suggesting evolutionary importance in viral replication and pathogenesis . Comparative sequence analysis reveals that while there are variations between different alphaherpesviruses (such as HSV-1, HSV-2, and pseudorabies virus), the functional domains responsible for trafficking and protein-protein interactions remain conserved. Notably, HSV US9 protein lacks lysine residues, a distinctive feature that impacts its post-translational modifications and potentially its stability .

What are the primary functional roles of US9 in HSV-2 infection?

US9 serves multiple critical functions during HSV-2 infection:

• Anterograde Transport: In conjunction with glycoprotein E/I (gE/gI), US9 promotes the anterograde transport of virus particles in neuronal axons, enabling the virus to spread from the neuronal cell body to axon terminals and ultimately to peripheral tissues .

• Viral Assembly: US9 contributes to cytoplasmic envelopment of viral particles through interactions with tegument proteins. Deletion of both gE and US9 blocks the assembly of enveloped particles in the neuronal cytoplasm, preventing virions from entering axons .

• Protein-Protein Interactions: US9 binds to multiple tegument proteins including UL16, VP22, UL11, and UL21, forming a complex network that facilitates viral assembly and transport .

• Viral Spread: US9 participates in viral spread across cell junctions, particularly in neurons, which is crucial for efficient viral dissemination without exposure to the extracellular environment and host immune responses .

What expression systems are most effective for producing recombinant HSV-2 US9?

The optimal expression system for recombinant HSV-2 US9 depends on the specific research objectives. For structural studies requiring proper protein folding and post-translational modifications, mammalian expression systems are preferable. HEK293T or Vero cells transfected with appropriate expression vectors can yield functionally relevant recombinant US9 protein.

For higher yield production, baculovirus-insect cell systems offer a compromise between proper eukaryotic protein processing and production scale. When using this system, it's essential to consider that US9 is ubiquitinated in its native form, and this modification may be important for functional studies .

When designing expression constructs, researchers should consider:

• Including epitope tags (His, FLAG, or EGFP) at positions that don't interfere with the protein's functional domains

• Preserving the native transmembrane domain if membrane association is important for the study

• Creating targeted mutations in trafficking motifs to study their specific roles in protein function

How can researchers effectively track US9 intracellular trafficking?

To investigate US9 intracellular trafficking, several complementary approaches can be employed:

• Fluorescent Protein Tagging: Enhanced green fluorescent protein (EGFP) fusion constructs allow real-time visualization of US9 trafficking in live cells. This approach has been successfully used to define the intracellular localization of US9 to the TGN region .

• Immunofluorescence with Confocal Microscopy: This technique enables co-localization studies with various cellular markers. For US9 trafficking studies, co-staining with TGN markers and endocytic pathway components is particularly informative .

• Internalization Assays: These assays can demonstrate the dynamic nature of US9 distribution. Researchers can use antibodies against the extracellular domain of US9 or against an epitope tag to track the protein's movement from the cell surface to intracellular compartments .

• Site-Directed Mutagenesis: Mutation of specific motifs (such as the dileucine endocytosis signal or acidic cluster) followed by trafficking analysis can identify sequences necessary for proper localization and movement .

What purification strategies yield the highest purity recombinant US9 protein?

Purification of recombinant US9 presents challenges due to its membrane association and various post-translational modifications. A multi-step purification strategy is recommended:

• Membrane Fraction Isolation: Begin with differential centrifugation to isolate membrane fractions containing US9.

• Detergent Solubilization: Use mild detergents (CHAPS, DDM, or Triton X-100) to solubilize US9 while maintaining its native conformation.

• Affinity Chromatography: If using tagged constructs (His-tag or FLAG-tag), employ the corresponding affinity resins for initial capture.

• Size Exclusion Chromatography: This step helps separate US9 from remaining contaminants and different oligomeric states.

• Ion Exchange Chromatography: As a final polishing step, particularly useful for removing nucleic acid contaminants.

For studies requiring native, untagged protein, consider using US9-specific antibodies for immunoaffinity purification. Always verify purity by SDS-PAGE and Western blotting, noting that US9 typically appears as multiple bands ranging from 12-25 kDa due to its post-translational modifications .

What techniques are most reliable for studying US9 interactions with tegument proteins?

Several complementary techniques provide robust data for studying US9 interactions with tegument proteins:

• Co-immunoprecipitation (Co-IP): This is the most widely used approach for detecting protein-protein interactions in HSV research. Studies have successfully used this technique to demonstrate that tegument proteins UL16, VP22, UL11, and UL21 interact with US9 in infected cells . When performing Co-IP:

  • Use appropriate detergents that maintain membrane protein interactions

  • Include both positive controls (known interactions) and negative controls (irrelevant antibodies)

  • Compare results from infected cells versus transfected cells expressing individual proteins to distinguish direct from indirect interactions

• Proximity Ligation Assay (PLA): This technique allows visualization of protein interactions in situ with high sensitivity and specificity, providing spatial information about where US9-tegument interactions occur within the cell.

• Yeast Two-Hybrid or Mammalian Two-Hybrid: These can identify direct binary interactions, though membrane proteins like US9 may present technical challenges.

• Bimolecular Fluorescence Complementation (BiFC): This approach not only confirms interactions but also reveals their subcellular localization.

• Pull-down Assays with Recombinant Proteins: Using purified components can confirm direct interactions and determine binding affinities.

Research has shown that interactions between US9 and tegument proteins appear stronger in infected cells compared to transfected cells, suggesting that a matrix of several different tegument proteins forms in infected cells that enhances binding to US9 .

How does ubiquitination affect US9 function and stability?

US9 exhibits an unusual property among viral proteins - it is ubiquitinated yet remains stable for extended periods. This apparent contradiction provides insights into non-canonical roles of ubiquitination:

Stability Profile: Despite ubiquitination, which typically targets proteins for degradation, US9 remains stable for at least 4 hours after entry into cells during infection . This stability is particularly interesting given that US9 lacks lysine residues, which are the conventional ubiquitination sites in proteins .

Functional Implications: The ubiquitination of US9 may serve roles beyond protein degradation:

• It might modulate protein-protein interactions with tegument components

• It could influence subcellular trafficking and localization

• It may play a role in viral assembly processes

Experimental Approaches: To study US9 ubiquitination, researchers can:

• Use proteasome inhibitors to assess whether ubiquitination affects protein turnover

• Employ mass spectrometry to identify the precise ubiquitination sites and types (K48 vs. K63 linkages)

• Generate ubiquitination-deficient mutants to assess the impact on viral replication and spread

• Investigate interactions between US9 and the proteasomal machinery, as evidence suggests US9 interacts with proteasome subunits

What is the significance of the acidic cluster in US9's cytoplasmic tail?

The acidic cluster in US9's cytoplasmic tail is a crucial functional motif with multiple roles:

• Endocytic Trafficking: Deletion analysis has demonstrated that this acidic cluster containing putative phosphorylation sites is necessary for recycling US9 from the plasma membrane. Without this cluster, US9 relocalizes to the plasma membrane due to defective endocytosis .

• TGN Retention: The acidic cluster contributes to the retention of US9 in the TGN region through its role in the retrieval pathway from the cell surface .

• Potential Phosphorylation: The cluster contains putative tyrosine and casein kinase I and II phosphorylation sites, suggesting regulation through phosphorylation events .

• Virion Incorporation: Interestingly, the acidic motif does not contain signals needed to direct the incorporation of US9 into viral envelopes. Mutants lacking this domain are still competent to be incorporated efficiently into virion envelopes, indicating separate mechanisms for envelope incorporation and trafficking .

To experimentally investigate this motif, site-directed mutagenesis of specific acidic residues or potential phosphorylation sites, followed by trafficking analysis and functional assays, can elucidate the contribution of individual amino acids within this cluster.

How can researchers effectively design US9 deletion or mutation studies to understand its role in neuronal transport?

Designing effective US9 deletion or mutation studies requires careful consideration of protein domains and cellular context:

Mutation Strategy Design:

• Domain-Specific Mutations: Target specific functional domains:

  • Acidic cluster mutations to disrupt endocytosis

  • Dileucine motif mutations to alter trafficking

  • Transmembrane domain modifications to assess membrane integration requirements

  • C-terminal modifications to examine tegument protein interactions

• Rescue Experiments: Always include complementation studies with wild-type US9 to confirm phenotype specificity.

• Double Mutant Analysis: Consider creating double mutants (e.g., US9/gE deletion) to examine potential redundant functions, as research shows deletion of both gE and US9 blocks the assembly of enveloped particles in neuronal cytoplasm .

Neuronal Model Selection:

• Primary Neurons: Dorsal root ganglia neurons or superior cervical ganglia provide physiologically relevant systems but are technically challenging.

• Neuronal Cell Lines: Differentiated PC12 cells, Neuro-2a, or SH-SY5Y cells offer reproducibility and ease of manipulation.

• Compartmentalized Culture Systems: Microfluidic devices or Campenot chambers allow separation of neuronal cell bodies from axon termini, enabling directional transport studies.

Analytical Methods:

• Live Cell Imaging: Using fluorescently tagged viral particles to track axonal transport in real-time.

• Biochemical Fractionation: Separate axonal from cell body compartments to quantify viral components.

• Electron Microscopy: To visualize enveloped particles in different neuronal compartments.

• Proximity-dependent Biotinylation: To identify US9 interaction partners specifically in neuronal contexts.

What are the optimal methods for analyzing US9's role in viral assembly and envelopment?

Analyzing US9's role in viral assembly and envelopment requires multifaceted approaches:

Ultrastructural Analysis:

• Transmission Electron Microscopy (TEM): The gold standard for visualizing viral assembly intermediates. Compare wild-type virus with US9 mutants to identify accumulation of specific assembly intermediates (e.g., non-enveloped capsids).

• Correlative Light and Electron Microscopy (CLEM): Combines fluorescence microscopy with TEM to correlate US9 localization with assembly events.

• Immuno-electron Microscopy: Using gold-labeled antibodies to precisely localize US9 during assembly.

Biochemical Approaches:

• Subcellular Fractionation: Isolate and characterize viral assembly intermediates from different cellular compartments.

• Protein Cross-linking: To capture transient interactions during assembly.

• Pulse-Chase Analysis: To track the kinetics of viral protein incorporation into virions.

Genetic Approaches:

• Complementation Analysis: Test which domains of US9 can rescue assembly defects.

• Temperature-sensitive Mutants: Allow temporal control over US9 function.

• Inducible Expression Systems: To examine US9's role at specific stages of infection.

Quantitative Metrics:

Research has shown that deletion of both US9 and gE affects the stability of tegument protein UL16 in neurons, suggesting a key role in the tegument-envelope interaction network critical for assembly .

How can researchers differentiate between the functions of US9 in different cell types, particularly neurons versus epithelial cells?

US9 exhibits cell-type specific functions, particularly in neurons. Effective experimental design to differentiate these functions includes:

Parallel Cell System Analysis:

• Matched Infection Conditions: Conduct parallel infections in neuronal and epithelial cells using identical viral strains and MOI.

• Viral Growth Kinetics: Compare multi-step growth curves between cell types with wild-type versus US9-mutant viruses.

• Cell-specific Markers: Monitor infection progression using cell-type specific markers alongside viral proteins.

Neuron-Specific Techniques:

• Microfluidic Chambers: These allow separation of neuronal cell bodies from axons, enabling assessment of directional transport.

• Compartmentalized Analysis: Quantify viral components in cell bodies versus axons to assess transport efficiency.

• Live-cell Imaging: Track labeled viral particles in real-time to measure transport dynamics and directionality.

Molecular and Biochemical Analysis:

• Interactome Comparison: Use proteomics to compare US9 binding partners in neurons versus epithelial cells.

• Post-translational Modification Analysis: Examine whether US9 is differentially modified in different cell types.

• Expression Level Quantification: Compare US9 expression kinetics and abundance between cell types.

Research indicates that US9 binding to tegument proteins has neuron-specific effects on HSV assembly, which is required for axonal transport of enveloped particles . In epithelial cells, deletion of US9 alone often has minimal effects on viral replication, whereas in neurons, it significantly impairs anterograde transport and spread.

How do researchers resolve contradictory findings regarding US9 function across different herpesvirus species?

Resolving contradictions in US9 research across herpesvirus species requires systematic approaches:

Sources of Variation:

• Evolutionary Divergence: Despite conservation, US9 proteins from different alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus, varicella-zoster virus) have acquired species-specific adaptations.

• Experimental Systems: Differences in cell types, viral strains, and methodologies contribute to apparent contradictions.

• Functional Redundancy: Varying degrees of functional overlap between US9 and other viral proteins (particularly gE/gI) across species.

Resolution Strategies:

• Direct Comparative Studies:

  • Use identical experimental conditions to compare US9 from different viruses

  • Create chimeric US9 proteins swapping domains between species

  • Conduct cross-complementation studies to assess functional equivalence

• Standardized Assays:

  • Develop quantitative, standardized assays for US9 functions

  • Establish clear metrics for phenotype assessment

  • Use multiple assays to measure each function

• Systems Biology Approaches:

  • Map complete interaction networks for US9 across species

  • Identify conserved versus species-specific interactions

  • Correlate interaction differences with functional variations

Interpretation Framework:

When interpreting contradictory findings, researchers should consider whether differences represent true biological variation or technical artifacts, and design experiments specifically to distinguish between these possibilities.

What are the best approaches for analyzing the dynamic relationship between US9 and the ubiquitin-proteasome system?

The unusual relationship between US9 and the ubiquitin-proteasome system—being ubiquitinated yet stable—presents intriguing research questions:

Key Experimental Approaches:

• Dynamic Ubiquitination Analysis:

  • Pulse-chase experiments combined with immunoprecipitation

  • Ubiquitin remnant profiling using mass spectrometry

  • Live-cell sensors for ubiquitination events

• Proteasomal Interaction Studies:

  • Co-immunoprecipitation with proteasome subunits under various conditions

  • Proximity labeling to identify dynamic interactions

  • In vitro reconstitution with purified components

• Functional Manipulation:

  • Proteasome inhibitors at various infection stages

  • Deubiquitinating enzyme (DUB) overexpression or inhibition

  • Targeted degradation approaches (e.g., dTAG system)

Specialized Techniques:

• Ubiquitin Chain Typing: Determine which ubiquitin linkage types (K48, K63, etc.) are present on US9, as this dictates functional outcomes.

• Single-molecule Tracking: Follow individual US9 molecules to assess their fate after ubiquitination.

• Correlation of Ubiquitination with Function: Assess how ubiquitination status correlates with specific US9 functions.

Interpretive Framework:
Research has shown that immune complexes consisting of proteasomal proteins and antibodies against proteasome subunits can pull down US9 protein . This suggests that despite being stable and lysine-less, US9 engages with the proteasomal machinery, potentially for non-degradative functions.

A working model suggests that US9's ubiquitination may serve regulatory rather than degradative functions, potentially influencing protein-protein interactions or trafficking. Ubiquitination may also represent a host defense mechanism that the virus has evolved to exploit.

How can researchers accurately distinguish direct versus indirect effects when studying US9 mutations on viral transport and assembly?

Distinguishing direct from indirect effects of US9 mutations presents significant challenges:

Experimental Design Strategies:

• Temporal Control Systems:

  • Inducible expression of wild-type US9 in mutant backgrounds

  • Temperature-sensitive US9 mutants

  • Rapid protein degradation systems (e.g., auxin-inducible degron)

• Spatial Localization Approaches:

  • Targeted US9 mislocalization (adding alternative targeting signals)

  • Anchoring US9 to specific compartments

  • Photoactivatable or optogenetic US9 variants

• Protein Engineering:

  • Interface-specific mutations based on structural data

  • Creation of separation-of-function mutants

  • Minimally disruptive tagging strategies

Analytical Approaches:

• Hierarchical Phenotyping:

  • Assess immediate biochemical consequences before examining downstream effects

  • Time-course studies to establish causality

  • Single-cell analyses to account for heterogeneity

• Interaction Network Mapping:

  • Compare wild-type and mutant US9 interactomes

  • Identify primary versus secondary changes in interaction networks

  • Correlation analysis between interaction changes and phenotypic effects

• Rescue Experiments:

  • Targeted complementation with specific interacting partners

  • Trans-complementation with minimal functional domains

  • Chemical rescue approaches where applicable

Interpretive Considerations:
Research has shown that coprecipitation of gE/gI and US9 was observed in HSV-infected cells but not in transfected cells, suggesting against direct US9-gE/gI interactions . Instead, a matrix of several different tegument proteins formed in infected cells appears to mediate these connections. This highlights the importance of distinguishing between direct protein-protein interactions and effects mediated through multi-protein complexes.