Recombinant Equine herpesvirus 1 Glycoprotein gp2 (EUs4)

What is EHV-1 gp2 and where is it encoded in the viral genome?

EHV-1 gp2 is a unique glycoprotein expressed from gene 71 (EUs4) located within the unique short (Us) region of the EHV-1 genome . Unlike most viral glycoproteins that have homologs across alphaherpesviruses, gp2 is found only in EHV-1, EHV-4, and asinine herpesvirus 3 . The glycoprotein is among the most abundant and immunogenic proteins in EHV-1 virions, making it a significant target for immune responses during infection .

To study this protein, researchers typically start with the genomic sequence. The EUs4 gene of pathogenic EHV-1 strain RacL11 is an open reading frame of 2,376 bp encoding a protein of 791 amino acids . This contrasts with the attenuated strain KyA, which harbors an in-frame deletion of 1,242 bp (from position 222 to 1461), resulting in a truncated protein of only 383 amino acids .

What are the structural characteristics of EHV-1 gp2?

EHV-1 gp2 is a heavily O-glycosylated protein with several notable structural features:

• The full-length protein in virulent strains like RacL11 is approximately 250 kDa in size

• It is rich in serine and threonine residues, making it highly susceptible to O-glycosylation

• The protein undergoes endoproteolytic cleavage at adjacent arginine residues (positions 506 and 507) in the sequence HRGRAGGR...506R507G

• This cleavage results in a 42-kDa carboxy-terminal subunit containing the transmembrane anchor and an N-terminal component that is highly O-glycosylated

• The molecular mass of native gp2 ranges from 192 to >400 kDa due to extensive glycosylation

This structural complexity contributes to the protein's functional roles in viral replication and pathogenesis, as well as its immunogenicity.

How does gp2 differ between virulent and attenuated EHV-1 strains?

The most significant difference between gp2 in virulent and attenuated strains is the presence of a large internal deletion in the attenuated KyA strain. This comparison is summarized in the table below:

This comparison demonstrates that the structural differences directly correlate with functional differences, particularly in virulence .

What methods are available for detecting EHV-1 gp2 in laboratory settings?

Several methods have been developed for detecting EHV-1 gp2 in research settings:

• Antibody-based detection: Monoclonal antibody 8B6 is directed against the terminal region of EHV-1 gp2 and recognizes both the full-length protein and the 42-kDa cleavage product . Commercial polyclonal antibodies are also available, such as those directed against the C-terminal residues of wild-type EHV-1 gp2 .

• Immunofluorescence assays: These can be performed using anti-gp2 antibodies to detect the protein in infected cells, often in combination with other markers like glial fibrillary acidic protein (GFAP) in cell culture models .

• Western blot analysis: This technique allows the detection of different molecular masses of gp2 and has revealed that horse sera against EHV-1 recognize different forms of the protein compared to monoclonal antibodies .

• Molecular detection: PCR assays targeting gene 71 can be designed for detecting the genomic sequence encoding gp2, which is particularly useful for differentiating between strains with full-length and truncated versions of the gene.

Each method has specific applications depending on the research question being addressed, with antibody-based techniques being particularly valuable for studying protein expression and localization.

How does the gp2 form affect virus growth and cell-to-cell spread?

Research has revealed significant differences in virus growth and cell-to-cell spread depending on the form of gp2 expressed:

• Absence of gp2 in RacL11 background:

  • 6-fold reduction in extracellular virus titers

  • 13% reduction in plaque diameters

• Absence of gp2 in KyA background:

  • 55% reduction in plaque diameter

  • 51-fold decrease in extracellular virus titers

• Complementation studies:

  • The growth defects of gp2-negative KyA could be restored by reinsertion of the truncated gp2 gene

  • Interestingly, insertion of full-length gp2 did not restore normal growth

This suggests that truncated and full-length gp2 are not functionally equivalent and cannot compensate for each other in allogeneic virus backgrounds . The molecular basis for these differences likely involves the role of gp2 in virus egress, as EHV-1 strains lacking gp2 are impaired in this process while secondary envelopment appears to occur with unaltered efficiency .

What is the molecular mechanism by which full-length gp2 contributes to EHV-1 virulence?

The full-length gp2 contributes to EHV-1 virulence primarily by inducing a robust inflammatory response in infected tissues. Studies comparing infections with KyA (truncated gp2) and KyARgp2F (expressing full-length gp2) revealed:

• Enhanced inflammatory cell recruitment:

  • Higher numbers of T and B lymphocytes in the lungs

  • Extensive consolidation with large numbers of Mac-1-positive cells

• Increased cytokine and chemokine expression:

  • RNase protection assays showed increased expression of numerous inflammatory mediators at 12 hours post-infection, including:

    • Cytokines: IL-1β, IL-6, TNF-α, IFN-γ

    • Chemokines: MIP-1α, MIP-1β, MIP-2, IP-10, MCP-1, TAG-3

• Genome-wide inflammatory response:

  • DNA array experiments confirmed a 2- to 13-fold increase in expression of 31 inflammatory genes at 8 and 12 hours post-infection with KyARgp2F compared to KyA

  • Additional upregulated factors included CXCL9, CCL7, CCL12, CXCL11, CCL20, CXCL1, and CCL11

• Pattern recognition receptor activation:

  • Higher levels of transcripts for TLR2, TLR3, and TLR6

  • Increased expression of chemokine receptors CCR1 and CCR2

This extensive inflammatory cascade triggered by full-length gp2 results in severe immunopathology and contributes significantly to disease severity.

How should experimental design approach the study of EHV-1 gp2 in pathogenesis?

When designing experiments to study the role of gp2 in EHV-1 pathogenesis, researchers should consider several methodological approaches:

• Recombinant virus construction:

  • Use bacterial artificial chromosome (BAC) cloning or transfection-based methods to generate virus mutants with specific alterations in the gp2 gene

  • Current approaches have produced various constructs as shown in this table from the literature:

• In vitro assays:

  • Compare virus growth kinetics in multiple cell types, including:

    • Equine dermis (NBL6)

    • Rabbit kidney (RK-13)

    • Human epithelial kidney (HEK293)

    • Mouse lung epithelial (MLE12)

  • Measure plaque formation, cell-to-cell spread, and extracellular virus release

• Animal models:

  • The CBA mouse model has proven particularly useful for studying EHV-1 respiratory virulence

  • BALB/c mice have also been used but appear less susceptible to EHV-1-mediated disease

  • Standard infection protocol: intranasal inoculation with 1.5 × 10^6 PFU/mouse

  • Key parameters to measure:

    • Body weight changes

    • Viral titers in lungs

    • Inflammatory cell infiltration

    • Cytokine/chemokine expression profiles

• Controls and variables:

  • Include appropriate virus strain controls (virulent and attenuated)

  • Test multiple virus doses

  • Consider both acute and long-term outcomes

  • Use age and sex-matched animals to reduce variability

• Statistical considerations:

  • Determine appropriate sample sizes using power analysis

  • Use statistical methods appropriate for the experimental design (e.g., repeated-measures ANOVA for weight loss over time)

Following these methodological approaches will ensure robust and reproducible results when studying the role of gp2 in EHV-1 pathogenesis.

What are the implications of gp2 research for EHV-1 vaccine development?

Research on EHV-1 gp2 has significant implications for vaccine development:

These findings underscore the complexity of using gp2 in vaccine development and highlight the need for careful design approaches that maximize immunogenicity while minimizing potential virulence.

How does post-translational processing of gp2 affect its function?

The post-translational processing of gp2 has significant implications for its structure and function:

• O-glycosylation:

  • The serine/threonine-rich nature of gp2 makes it a target for extensive O-glycosylation

  • This modification increases the molecular mass of the protein from its predicted size to 192-400 kDa

  • The glycosylation pattern likely affects protein folding, stability, and interactions with host factors

• Endoproteolytic cleavage:

  • EHV-1 gp2 undergoes cleavage after adjacent arginine residues 506 and 507

  • This processing generates:

    • A 42-kDa C-terminal subunit containing the transmembrane domain

    • A heavily glycosylated N-terminal component

  • Both the full-length precursor and the cleaved products are incorporated into virus particles

• Functional implications:

  • The truncated gp2 in KyA (383 amino acids) lacks a significant portion of the protein but retains the C-terminal region

  • This truncated form appears to function differently in virus growth and spread

  • In KyA, the truncated gp2 can restore growth defects of gp2-negative virus, but the full-length gp2 cannot

  • This suggests that processing of the protein is finely tuned to its functional role

• Interspecies differences:

  • Unlike EHV-1 gp2, the EHV-4 homolog is not cleaved into two polypeptides in infected cells

  • This difference may contribute to the distinct biological properties of these related viruses

Understanding these processing events is crucial for developing recombinant forms of gp2 for research and vaccine applications, as proper folding and modification will be essential for maintaining native functions.

How does gp2 compare with glycoproteins from other herpesviruses?

Comparative analysis of gp2 with glycoproteins from other herpesviruses reveals several important distinctions:

• Limited conservation:

  • gp2 homologs are found only in EHV-1, EHV-4, and asinine herpesvirus 3

  • This contrasts with other herpesvirus glycoproteins like gB, gC, gD, etc., which are conserved across alphaherpesviruses

• Functional differences:

  • Many conserved herpesvirus glycoproteins (like gB, gD) are essential for virus entry

  • In contrast, gp2 appears to function primarily in virus egress and cell-to-cell spread

  • gp2 is not essential for virus growth in cell culture but significantly impacts virus titers and spread

• Compensatory mechanisms:

  • The attenuated KyA strain lacks both the gE-gI complex and contains truncated gp2

  • Research suggests that truncation of gp2 may have compensated for the absence of gE and gI, allowing KyA to overcome growth restrictions in cultured cells

  • This indicates potential functional overlap or compensation between different viral glycoproteins

• Structural uniqueness:

  • The heavy O-glycosylation and specific cleavage pattern of gp2 make it structurally distinct from many other herpesvirus glycoproteins

  • While many herpesvirus glycoproteins form heterodimers or heterooligomers (like gE-gI or gH-gL), gp2 appears to function independently

• Virulence contribution:

  • Full-length gp2 makes a significant contribution to respiratory virulence in EHV-1

  • This specific virulence mechanism through inflammatory pathway activation represents a unique feature not shared by all herpesvirus glycoproteins

This comparative perspective helps researchers understand the specific contributions of gp2 to EHV-1 biology and may inform approaches to targeting this protein for therapeutic or preventive interventions.