Recombinant Suid herpesvirus 1 Transcriptional regulator IE63 homolog (UL54)

What is the Suid herpesvirus 1 Transcriptional regulator IE63 homolog (UL54)?

UL54 is a multifunctional protein encoded by Pseudorabies virus (PRV), also known as Suid herpesvirus 1. This protein plays critical roles in shutoff of host protein synthesis, transactivation of virus and cellular genes, and regulation of splicing and translation. UL54 shares significant homology with immediate-early regulatory proteins in other herpesviruses, particularly with ICP27 from Herpes Simplex Virus type 1 (HSV-1) and open reading frame 4 (ORF4) from Varicella-Zoster Virus (VZV) .

The genetic characterization of UL54 has revealed that despite its important regulatory functions, the gene is surprisingly dispensable for PRV growth in tissue culture, though its deletion results in a small-plaque phenotype and altered viral gene expression patterns. These altered phenotypes can be complemented in trans by both HSV-1 ICP27 and VZV ORF4 proteins, demonstrating functional conservation across herpesvirus homologs .

How does UL54 affect viral gene expression?

UL54 exhibits significant regulatory effects on PRV gene expression through multiple mechanisms. Studies with UL54-null mutants (vJSΔ54) have revealed that UL54 deletion differentially affects viral protein expression patterns. Specifically, the glycoprotein gC accumulates to lower levels in cells infected with vJSΔ54 compared to wild-type virus, while gK levels become undetectable. Conversely, other late gene products, including gB, gE, and Us9, accumulate to higher levels than in wild-type virus infections in a multiplicity-dependent manner .

At the transcriptional level, UL54 appears to regulate UL53 and UL52 genes, as their respective RNA levels are decreased in cells infected with UL54-deficient viruses. This indicates that UL54 functions as a transcriptional regulator for at least a subset of viral genes. Additionally, DNA replication is reduced in cells infected with vJSΔ54, suggesting that UL54 may either directly or indirectly influence viral DNA synthesis .

What is the relationship between UL54 and host protein synthesis?

Unlike some of its herpesvirus homologs that are involved in host shutoff, deletion of UL54 in PRV (vJSΔ54) had no effect on the ability of the virus to shut off host cell protein synthesis. This finding suggests that in PRV, UL54 does not play a significant role in the suppression of host protein production, and that this function is likely mediated by other viral factors. This represents an important difference between UL54 and some of its homologs in other herpesviruses, where they may contribute to host shutoff mechanisms .

The maintenance of host shutoff capabilities in UL54-deficient PRV indicates that the protein's primary functions are more likely related to viral gene regulation rather than direct antagonism of host protein synthesis. This also suggests that researchers studying host-pathogen interactions should focus on other viral factors when investigating PRV-mediated host shutoff mechanisms .

What methodologies are most effective for generating UL54 null mutations?

The generation of UL54 null mutations in PRV can be accomplished through several molecular approaches, with bacterial artificial chromosome (BAC) systems offering significant advantages over traditional homologous recombination in mammalian cells. Two particularly effective methodologies have been documented:

• Sugar Suicide System for Allele Exchange: This approach involves constructing homology regions flanking the UL54 locus and a kanamycin resistance determinant. The methodology requires amplifying approximately 300 bp of homology regions upstream of the UL54 start site and the last 252 bp of the UL54 ORF plus 48 bp downstream of the stop codon. These regions are joined by PCR and cloned into appropriate vectors (such as pGEM-5zf(+)). After insertion of a kanamycin resistance cassette, the construct is transferred to an allele exchange vector (such as pGS284) for recombination with the PRV BAC in E. coli .

• λRed Recombineering System: This more efficient system requires minimal homology (as little as 30-64 bp) to promote allele exchange. The procedure involves:

  • Creating a targeting cassette with the RpsL-Neo selection-counterselection system flanked by UL54 homology regions

  • Performing a precise insertion using the λRed system

  • Conducting a subsequent "loop-out" step using a 140-bp PCR product with appropriate homology regions to remove the selection markers and the remaining UL54 sequence

The λRed system offers advantages in precision, efficiency, and the ability to create clean deletions with minimal disruption to surrounding genomic regions. For verification of successful UL54 deletion, Southern blot analysis using appropriate probes and restriction enzyme digestion patterns has proven effective .

How does UL54 deletion affect viral pathogenesis in animal models?

UL54 deletion significantly attenuates PRV pathogenesis in animal models. In a mouse model of PRV infection, animals infected with the UL54-null virus (vJSΔ54) demonstrate substantially extended survival times, living approximately twice as long as those infected with wild-type virus. This attenuation correlates with delayed accumulation of virus-specific antigens in key tissues, including skin, dorsal root ganglia, and spinal cord .

The specific mechanisms underlying this attenuation likely involve multiple aspects of UL54 function. The reduced viral DNA replication and altered gene expression patterns observed in vitro may contribute to the reduced pathogenicity in vivo. Additionally, the regulatory effects of UL54 on viral genes like UL53 and UL52 may impact viral spread, neuroinvasion, or immune evasion capabilities .

For researchers investigating viral attenuation strategies, UL54 represents a promising target for developing attenuated vaccine strains or understanding fundamental aspects of alphaherpesvirus pathogenesis. The significant attenuation without complete abolishment of viral replication makes UL54-null viruses potentially valuable vaccine vectors or oncolytic agents .

What experimental approaches can determine the precise transcriptional targets of UL54?

To elucidate the specific transcriptional targets regulated by UL54, researchers should consider implementing a multi-faceted experimental strategy:

• RNA-Seq Comparative Analysis: Comparing transcriptomes of cells infected with wild-type versus UL54-null PRV at multiple time points post-infection can provide a comprehensive view of genes differentially regulated by UL54. This approach could reveal both viral and cellular transcriptional targets.

• Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq): Using antibodies against UL54 or epitope-tagged versions of the protein, researchers can identify genomic regions directly bound by UL54, providing evidence for direct transcriptional regulation.

• Promoter-Reporter Assays: Cloning viral promoters (particularly those of UL53 and UL52, which show reduced expression in UL54-null viruses) upstream of reporter genes can help quantify UL54's direct effects on transcriptional activation or repression .

• RNA Immunoprecipitation (RIP): Since UL54 may also function post-transcriptionally in RNA processing or stability, RIP experiments can identify RNA molecules directly interacting with the protein.

• CRISPR-Cas9 Screening: Systematic modification of potential UL54 binding sites in viral promoters can help map the specific DNA elements required for UL54-mediated regulation.

How do the functional domains of UL54 contribute to its regulatory activities?

The functional domains of UL54 and its contributions to regulatory activities can be investigated through several experimental approaches:

• Domain Mapping through Mutagenesis: Creating a series of deletion or point mutants targeting conserved domains can help identify regions essential for specific functions. Based on homology with other herpesvirus counterparts, the C-terminal region is likely to contain critical functional domains, as this is the most highly conserved region across UL54 homologs .

• Protein-Protein Interaction Studies: Techniques such as co-immunoprecipitation, yeast two-hybrid assays, or proximity-dependent biotin identification (BioID) can identify viral and cellular proteins interacting with UL54, potentially revealing how these interactions mediate its regulatory functions.

• Subcellular Localization Analysis: Immunofluorescence microscopy or fractionation studies can determine where UL54 localizes within infected cells during different stages of viral replication, providing insights into its potential functional roles.

• In vitro Biochemical Assays: Purified recombinant UL54 can be tested for specific activities such as RNA binding, DNA binding, or effects on transcription using cell-free systems.

The fact that UL54 can be complemented in trans by both HSV-1 ICP27 and VZV ORF4 proteins suggests functional conservation of key domains across herpesvirus homologs. This provides an opportunity for comparative studies to identify essential structural elements shared among these proteins .

What are the molecular mechanisms underlying UL54's effects on DNA replication?

The observed reduction in DNA replication in cells infected with UL54-null viruses suggests that UL54 influences viral DNA synthesis, either directly or indirectly. Several experimental approaches can elucidate the underlying mechanisms:

• Quantitative Analysis of Replication Fork Progression: Techniques such as DNA fiber analysis or 2D gel electrophoresis can determine if UL54 affects the initiation, elongation, or termination phases of DNA replication.

• Analysis of Replication Protein Expression: Quantitative assessment of levels and post-translational modifications of key replication proteins (including UL52, which shows reduced RNA levels in UL54-null infections) can reveal if UL54 indirectly affects DNA replication through regulation of replication machinery components .

• Chromatin Immunoprecipitation of Replication Factors: ChIP assays targeting viral replication proteins can determine if UL54 deletion affects their recruitment to viral origins of replication.

• Cell Cycle Analysis: Since herpesvirus replication interactions with cellular factors are often cell-cycle dependent, investigating whether UL54 affects cell cycle progression in infected cells may provide insights into its role in DNA replication.

The transcriptional regulation of UL52 by UL54 is particularly significant, as UL52 encodes a component of the helicase-primase complex essential for viral DNA replication. This suggests that at least part of UL54's effect on DNA replication may be mediated through its regulation of UL52 expression .

What verification methods confirm successful generation of UL54-null viruses?

Verification of UL54-null viruses requires a comprehensive approach combining genomic, transcriptomic, and proteomic analyses:

• Southern Blot Analysis: This remains the gold standard for confirming genomic alterations. For UL54 deletions, appropriate probes should target both the deleted region and flanking sequences. Expected restriction fragment pattern changes include:

  • In vJSΔ54, the 5′ BamHI fragment shifts from ~7.4 kb to ~4.9 kb

  • Loss of specific SalI and NcoI fragments, depending on the exact deletion construct

• PCR Verification: Primers flanking the deletion site can amplify products of predictable sizes that differ between wild-type and mutant viruses. Sequence analysis of these PCR products provides definitive confirmation of the genetic arrangement.

• Northern Blot or RT-PCR: These methods verify the absence of UL54 transcripts in cells infected with deletion mutants.

• Western Blot Analysis: Using antibodies specific to UL54 protein confirms the absence of protein expression in cells infected with deletion mutants.

• Functional Complementation: Testing whether the small-plaque phenotype can be complemented by expression of UL54 homologs (such as HSV-1 ICP27 or VZV ORF4) provides functional verification of the deletion .

For researchers creating UL54-null viruses, it is crucial to ensure that deletion strategies do not inadvertently affect adjacent genes or regulatory elements, particularly given that UL54 is 3′ coterminal with two upstream genes (UL52 and UL53) .

How can researchers effectively study UL54's role in viral replication kinetics?

To comprehensively assess UL54's impact on viral replication kinetics, researchers should implement multiple complementary approaches:

• Multi-step Growth Curves: Infecting cells with wild-type and UL54-null viruses at low multiplicity of infection (MOI) allows for measurement of virus production over multiple rounds of replication. Samples should be collected at regular intervals (e.g., every 4-6 hours) for at least 36-48 hours post-infection.

• Single-step Growth Curves: High MOI infections synchronized across all cells can isolate specific phases of the replication cycle where UL54 exerts its effects.

• Plaque Size Analysis: Quantitative measurement of plaque sizes provides insights into cell-to-cell spread capabilities of UL54-null viruses compared to wild-type .

• Quantitative PCR for Viral DNA: This allows precise quantification of viral genome replication over time, helping distinguish between defects in DNA replication versus other aspects of the viral life cycle.

• Time-of-Addition Studies with Complementing Proteins: Adding back UL54 or its homologs at different times post-infection can help define the temporal requirements for UL54 function.

Data should be analyzed using appropriate statistical methods, and experiments should include biological replicates across different cell types to determine if UL54's effects on replication are cell-type specific. The small-plaque phenotype of UL54-null viruses suggests particular attention should be paid to cell-to-cell spread mechanisms .

What animal models are most appropriate for studying UL54's role in pathogenesis?

The selection of appropriate animal models for studying UL54's role in pathogenesis should consider both the natural host range of PRV and experimental accessibility:

• Mouse Models: Mice have been successfully used to demonstrate that UL54-null PRV is highly attenuated, with infected animals surviving approximately twice as long as those infected with wild-type virus. These models allow for detailed analysis of viral antigen accumulation in tissues including skin, dorsal root ganglia, and spinal cord .

• Swine Models: As the natural host of PRV, pigs provide the most physiologically relevant system for studying pathogenesis. In swine, researchers can assess:

  • Clinical signs development and severity

  • Viral shedding and transmission

  • Tissue tropism and spread to the nervous system

  • Immune responses to infection

• Ex Vivo Tissue Systems: Organotypic cultures of porcine trigeminal ganglia or brain slices provide intermediate complexity systems to study neuroinvasion and spread without requiring whole animal experiments.

For meaningful results, researchers should consider:

• Standardizing viral doses and routes of administration

• Including both clinical scoring and molecular analyses of viral spread

• Incorporating immunohistochemistry to track virus-specific antigen accumulation in tissues

• Monitoring immune responses, particularly in natural host systems

The significantly longer survival time of animals infected with UL54-null virus makes this mutant particularly valuable for studying the molecular basis of virulence attenuation and potentially for developing attenuated vaccine candidates .

How might UL54's functions be leveraged for development of attenuated vaccines?

The significant attenuation of UL54-null PRV in animal models presents a compelling opportunity for vaccine development. Researchers pursuing this direction should consider:

• Safety Profiling: Comprehensive assessment of UL54-null viruses for residual virulence, potential for reversion, and safety in immunocompromised hosts is essential. The extended survival time of mice infected with vJSΔ54 suggests promising attenuation, but thorough evaluation in natural host species is necessary .

• Immunogenicity Assessment: Determine whether UL54-null viruses maintain sufficient immunogenicity despite attenuation. This includes measuring:

  • Humoral immune responses (neutralizing antibody titers)

  • Cell-mediated immunity (T cell responses to viral antigens)

  • Duration of protective immunity following vaccination

  • Cross-protection against heterologous PRV strains

• Genetic Stability Evaluation: Long-term passage studies to confirm that UL54-null viruses maintain their attenuated phenotype without compensatory mutations arising.

• Combination with Other Attenuated Mutations: Exploring whether combining UL54 deletion with modifications in other virulence genes might provide optimal balance between attenuation and immunogenicity.

• DIVA Capabilities: Assess whether UL54-null viruses could serve as DIVA (Differentiating Infected from Vaccinated Animals) vaccines, which is particularly important for PRV control programs.

The finding that UL54-null viruses delay accumulation of virus-specific antigens in tissues suggests they might be particularly suitable for generating attenuated vaccines with reduced risk of establishing latency while still eliciting protective immunity .

How do the functions of UL54 compare across different herpesvirus species?

Comparative analysis of UL54 homologs across herpesvirus species can provide valuable insights into conserved functions and species-specific adaptations:

• Functional Complementation Studies: The finding that both HSV-1 ICP27 and VZV ORF4 can complement the small-plaque phenotype of UL54-null PRV suggests significant functional conservation. Expanding these studies to include homologs from other herpesviruses could define the core conserved functions .

• Sequence-Structure-Function Analysis: Bioinformatic approaches combining sequence alignment, structural modeling, and functional data can identify:

  • Universally conserved domains likely essential for core functions

  • Lineage-specific modifications potentially related to host adaptation

  • Correlations between structural features and functional specializations

• Host-Range Determination: Investigating whether UL54 or its homologs contribute to viral host range restrictions by comparing their activities in cells from different species.

• Evolution Rate Analysis: Examining the evolutionary rates of different domains within UL54 homologs can reveal regions under selective pressure, potentially indicating host-pathogen interaction interfaces.

The variability in UL54 functions across herpesviruses is exemplified by the finding that while some homologs (like HSV-1 ICP27) are essential for viral replication, PRV UL54 is dispensable for growth in tissue culture despite affecting viral gene expression patterns .

What techniques can identify novel interaction partners of UL54?

Identifying UL54's interaction partners is crucial for understanding its molecular mechanisms. Researchers should consider these advanced methodologies:

• Proximity-dependent Biotin Identification (BioID): Fusion of UL54 with a promiscuous biotin ligase allows identification of proteins in close proximity during infection, potentially revealing transient or context-dependent interactions.

• Cross-linking Mass Spectrometry (XL-MS): Chemical cross-linking of protein complexes followed by mass spectrometry can capture direct interaction partners and provide structural information about interaction interfaces.

• Co-immunoprecipitation coupled with Tandem Mass Spectrometry: This approach can identify stable protein complexes containing UL54 at different stages of infection.

• RNA-Protein Interaction Analysis: Since UL54 homologs often interact with RNA, techniques such as CLIP-seq (Cross-linking Immunoprecipitation followed by sequencing) can identify RNA targets directly bound by UL54.

• Yeast Two-hybrid Screening: While more traditional, comprehensive library screening can uncover potential interactions that might be missed in infection-based systems.

• Mammalian Two-hybrid or Split-Luciferase Complementation Assays: These approaches allow verification of interactions in mammalian cells and assessment of interaction dynamics.

Researchers should focus particularly on potential interactions with components of the transcriptional machinery, RNA processing factors, and viral proteins involved in DNA replication, given UL54's known effects on these processes .