Recombinant Psittacid herpesvirus 1 Putative tegument protein UL11 (UL11)

What is the structural composition of UL11 in Psittacid herpesvirus 1?

UL11 in Psittacid herpesvirus 1 belongs to the conserved herpesvirus tegument protein family. Studies have revealed that UL11 is an intrinsically disordered protein (IDP) that undergoes liquid-liquid phase separation (LLPS) in vitro . Through techniques including circular dichroism, limited proteolysis, small-angle X-ray scattering (SAXS), and light microscopy, researchers have characterized its structural properties .

The protein contains:

• A C-terminal region with a disordered structure

• Fatty acyl modification sites for myristate and palmitate that mediate membrane binding

• An acidic cluster and dileucine motif important for recycling from plasma membrane to Golgi apparatus

Methodological approach: To characterize UL11 structure, employ a combination of:

How is recombinant PsHV-1 UL11 protein typically expressed and purified for experimental studies?

Expression and purification of recombinant PsHV-1 UL11 involves several methodological considerations:

Expression Systems:

• Bacterial expression using E. coli (BL21 or similar strains) with vectors like pET or pGEX systems

• For studying acylation and post-translational modifications, mammalian expression systems using HEK293T or chicken embryo fibroblasts (CEF) are preferred

Purification Protocol:

• For bacterial expression: Use affinity tags (His, GST) followed by size exclusion chromatography

• For mammalian expression: Concentrate virions from infected cell monolayers showing 90-95% cytopathic effect using PEG 8000 precipitation and pelleting through 30% sucrose cushion

• Extract proteins using 10% sodium dodecyl sulfate and proteinase K treatment

• For interaction studies: Use GST-pull down assays or co-immunoprecipitation methods to study binding partners

For optimal expression, culture conditions must be optimized to account for UL11's disordered nature, potentially using specialized media formulations like DMEM/F-12 supplemented with penicillin (50 μg/ml), streptomycin (50 μg/ml), and 10% fetal bovine serum .

What are the established cellular localization patterns of UL11 during PsHV-1 infection?

UL11 demonstrates dynamic localization during the viral replication cycle:

• Primarily accumulates on the cytoplasmic face of internal membranes

• Cycles between the plasma membrane and Golgi apparatus

• Trafficking to the Golgi apparatus is mediated by an acidic cluster and dileucine motif in UL11

• Co-localizes with UL16 in a Golgi-like compartment when co-expressed

Methodological approach for studying localization:

• Construct fluorescently tagged UL11 (GFP, mCherry) or use epitope tags (HA, FLAG)

• Perform immunofluorescence microscopy with markers for cellular compartments

• Use time-lapse microscopy to track trafficking patterns

• Employ subcellular fractionation with Western blotting to confirm localization

• Perform co-localization studies with Golgi markers and other viral proteins

The localization pattern is consistent across different alpha-herpesviruses, suggesting evolutionary conservation of UL11 trafficking mechanisms .

How does UL11 contribute to the secondary envelopment process in herpesvirus replication?

UL11 plays a critical role in secondary envelopment, as demonstrated by ultrastructural analyses of UL11-deleted mutants:

Mechanistic contributions:

• Forms a critical bridge between nucleocapsids and envelopment membranes

• Interacts with multiple viral proteins, particularly UL16, forming part of a protein network essential for envelopment

• May facilitate the recruitment of cellular ESCRT machinery to envelopment sites

• Deletion of UL11 homologs results in unenveloped capsid accumulation in the cytoplasm

Experimental evidence from comparative studies:
In Bovine herpesvirus 1 (BoHV-1), deletion of UL21 (which interacts with UL16, a UL11 binding partner) resulted in:

• 1,000-fold lower replication

• 85% smaller plaque size

• Accumulation of unenveloped capsids in the cytoplasm

Methodological approach for investigating envelopment:

• Generate UL11-deletion mutants using BAC mutagenesis

• Perform transmission electron microscopy to visualize envelopment defects

• Quantify cytoplasmic versus enveloped virions

• Use correlative light and electron microscopy to track UL11-tagged particles during envelopment

What protein-protein interaction network does UL11 establish during viral replication, and how can these interactions be experimentally validated?

UL11 establishes a complex interaction network essential for viral assembly:

Key interaction partners:

• UL16: Forms a stable complex with UL11; this interaction is conserved across alpha-herpesviruses

• UL21: Forms part of a potential UL11-UL16-UL21 complex

• Envelope glycoproteins: Potential interactions with gE

• Capsid-associated proteins: Suggested interactions to bridge capsid and envelope

Experimental validation methods:

• Co-immunoprecipitation (Co-IP):

  • Express tagged versions of UL11 (HA-tag) and potential partners

  • Pull down with appropriate antibodies

  • Analyze by Western blotting

• GST pull-down assays:

  • Express UL11 as GST fusion protein

  • Incubate with infected cell lysates

  • Identify binding partners by mass spectrometry

• Yeast two-hybrid screening:

  • Use UL11 as bait to screen for viral and cellular interactors

  • Validate hits with orthogonal methods

• Proximity labeling techniques:

  • BioID or APEX2 fusions to identify proteins in close proximity

  • Mass spectrometry identification of labeled proteins

• Förster Resonance Energy Transfer (FRET):

  • Tag UL11 and interacting partners with appropriate fluorophores

  • Measure energy transfer to confirm direct interactions

The UL11-UL16 interaction is particularly significant as UL11 homologs from pseudorabies and Marek's disease herpesviruses can also bind to HSV-1 UL16, suggesting evolutionary conservation of this interaction .

What is known about the intrinsically disordered nature of UL11 and its implications for liquid-liquid phase separation during viral assembly?

Recent research has revealed that UL11 belongs to a class of intrinsically disordered proteins (IDPs) with unique biophysical properties:

Characteristics and evidence:

• UL11 undergoes liquid-liquid phase separation (LLPS) in vitro

• The C-terminus has a disordered structure that fails to bind ribosomal RNA

• This disorder is found in multiple tegument proteins, suggesting a general assembly mechanism

Functional implications:

• LLPS may facilitate the concentration of viral components during assembly

• Disordered regions provide conformational flexibility for multiple interactions

• May contribute to the formation of viral assembly compartments as biomolecular condensates

Methodological approaches for studying LLPS and disorder:

• In vitro phase separation assays:

  • Observe condensate formation under various conditions

  • Test effects of salt, pH, and RNA on phase behavior

• Disorder prediction and validation:

  • Use computational tools (PONDR, IUPred) to predict disorder

  • Validate with CD spectroscopy and NMR

• Mutagenesis of disordered regions:

  • Generate variants with altered disorder propensity

  • Assess impact on viral assembly and replication

• Live-cell imaging of condensate formation:

  • Express fluorescently tagged UL11

  • Track formation of puncta during infection

Table: Comparative analysis of disorder characteristics across herpesvirus tegument proteins

How do mutations in the acidic cluster and dileucine motif of UL11 affect its function in viral replication and protein interactions?

The acidic cluster and dileucine motif of UL11 serve as critical functional domains:

Effects of mutations:

• Disruption of the acidic cluster and dileucine motif prevents association with the 40 kDa protein (UL16)

• These motifs are essential for recycling of UL11 from the plasma membrane to the Golgi apparatus

• Mutations affect trafficking but not necessarily membrane binding, which is primarily mediated by fatty acylation

Experimental design for mutational analysis:

• Generate a panel of UL11 mutants with:

  • Deletions or substitutions in the acidic cluster

  • Alterations in the dileucine motif

  • Combinations of both modifications

• Assess each mutant for:

  • Ability to bind UL16 via co-immunoprecipitation

  • Subcellular localization using immunofluorescence

  • Complementation of UL11-null virus replication

  • Impact on secondary envelopment via electron microscopy

• Quantitative measurements:

  • Binding affinities using surface plasmon resonance

  • Trafficking kinetics using live-cell imaging

  • Viral growth curves and plaque size analyses

Research findings:
Mutational studies revealed that while the entire second half of UL11 is not required for UL16 binding, the acidic cluster and dileucine motif are essential for this interaction . These motifs likely form a recognition surface that mediates both protein trafficking and establishment of the viral tegument protein network.

What methodologies can be employed to study the role of UL11 in cell-to-cell transmission of herpesviruses?

Cell-to-cell transmission is a critical aspect of herpesvirus pathogenesis and UL11 plays an essential role in this process:

Experimental approaches:

• Co-culture systems:

  • Infect donor cells with wild-type or UL11-mutant viruses

  • Co-culture with uninfected target cells

  • Use flow cytometry to quantify newly infected cells

• Neutralizing antibody assays:

  • Add neutralizing antibodies to block cell-free virus

  • Compare wild-type versus UL11-deficient virus spread

  • Results from related studies show cell-cell spread can occur despite presence of neutralizing antibodies

• Live-cell imaging:

  • Express fluorescently tagged viral proteins

  • Track virological synapse formation between cells

  • Monitor virus movement across cell junctions

• Virological synapse characterization:

  • Immunostain for UL11 at cell-cell contacts

  • Perform super-resolution microscopy to visualize synapse architecture

  • Use electron microscopy to examine ultrastructural features

• Genome delivery quantification:

  • Use qPCR to measure viral genome delivery to recipient cells

  • Compare cell-free versus cell-cell transmission efficiency

  • Data from related studies show higher number of genomes delivered via cell-cell route

Data from related research:
In studies of HCMV (which has the UL99 homolog of UL11), cell-to-cell transmission was shown to deliver larger numbers of viral genomes compared to cell-free infection, and this process occurred despite the presence of neutralizing antibodies .

What differences exist in UL11 structure and function across different herpesvirus subfamilies, and how can these be leveraged for comparative studies?

UL11 is conserved across herpesvirus subfamilies but exhibits important structural and functional variations:

Comparative features:

Research approaches for comparative studies:

• Sequence alignment and phylogenetic analysis:

  • Identify conserved versus divergent regions

  • Map functional domains across viral species

  • Use computational methods to predict structural conservation

• Complementation assays:

  • Express UL11 homologs from different viruses in UL11-null mutants

  • Assess functional complementation across species

  • Determine which features are universally conserved

• Domain swapping experiments:

  • Create chimeric UL11 proteins with domains from different viral species

  • Test functionality in various assays

  • Identify minimal functional domains

• Interactome comparison:

  • Characterize protein interaction networks for each UL11 homolog

  • Identify conserved versus species-specific interactions

  • Use proteomics approaches to map comprehensive interactomes

Studies have shown that UL11 homologs from pseudorabies and Marek's disease herpesviruses can interact with UL16 from HSV-1, indicating functional conservation despite sequence divergence . In beta-herpesviruses, the UL99 homolog has been determined to be an important factor in antiviral therapy research .

How can CRISPR-Cas9 genome editing be utilized to investigate UL11 function in the context of complete viral genomes?

CRISPR-Cas9 technology offers powerful approaches for studying UL11 function in herpesvirus biology:

Methodological strategies:

• Bacterial Artificial Chromosome (BAC) editing:

  • Design sgRNAs targeting UL11 in viral BAC

  • Create precise deletions, insertions, or point mutations

  • Reconstitute recombinant virus through transfection

  • This approach was successfully used to create a UL21 deletion mutant and revertant in BoHV-1

• In situ viral genome editing:

  • Deliver CRISPR-Cas9 components to virus-infected cells

  • Target UL11 in replicating viral genomes

  • Use HDR templates to insert reporters or tags

• Conditional knockout strategies:

  • Create destabilization domain-tagged UL11 variants

  • Enable conditional protein degradation to study timing effects

  • Use inducible promoters to control UL11 expression levels

• High-throughput screening:

  • Design CRISPR library targeting multiple regions of UL11

  • Screen for phenotypes related to viral assembly and spread

  • Identify critical residues through deep mutational scanning

Experimental design considerations:

• Include proper controls (wild-type and revertant viruses)

• Verify editing by sequencing and protein expression analysis

• Perform complementation assays with ectopically expressed UL11

• Utilize multiple cell types to assess cell-type specific functions

Expected outcomes:
UL11 deletion would likely result in significant replication defects, particularly in cell-to-cell spread and secondary envelopment, as observed with the UL21 deletion in BoHV-1 which showed 1,000-fold lower replication and 85% smaller plaque size .