Recombinant Alcelaphine herpesvirus 1 Putative C-type lectin protein A7 (A7)

What is the A7 protein of Alcelaphine herpesvirus 1?

The A7 protein of AlHV-1 is a type II glycoprotein containing a C-type lectin-like domain. It is a positional ortholog of the Epstein-Barr virus (EBV) glycoprotein gp42, encoded by the BZLF2 gene . The A7 gene is present in the pathogenic C500 strain of AlHV-1 but is absent in the attenuated WC11 strain due to a major genomic deletion . This protein is expressed on the viral envelope and plays a critical role in mediating cell-to-cell viral spread, particularly in certain cell types, which is essential for viral pathogenesis and the development of MCF in susceptible hosts .

How is A7 structurally and functionally related to other herpesvirus proteins?

A7 is a positional ortholog of the Epstein-Barr virus glycoprotein gp42, which belongs to the C-type lectin family . Like its EBV counterpart, A7 appears to be involved in regulating viral cell tropism and mediating viral entry into specific cell types . The protein contains a C-type lectin-like domain characteristic of proteins that bind carbohydrates in a calcium-dependent manner . This structural feature suggests A7 may function in receptor binding during the viral entry process, potentially recognizing specific surface markers on target cells.

What phenotypic differences are observed between AlHV-1 strains expressing or lacking A7?

The phenotypic differences between AlHV-1 strains expressing or lacking A7 are significant and cell-type dependent:

What is the molecular mechanism by which A7 regulates cell-to-cell spread versus cell-free propagation?

The molecular mechanism underlying A7's regulation of viral spread appears to involve a balance between cell-associated and cell-free viral propagation. Experimental evidence indicates that A7 promotes cell-to-cell spread while simultaneously limiting cell-free viral dissemination .

When A7 is present (as in the C500 strain), the virus preferentially spreads through direct cell-to-cell contact, forming syncytia in susceptible cell types . This is evidenced by experiments using carboxymethylcellulose (CMC), which inhibits the release of infectious virions into the medium. When CMC was applied to A7STOP-207 infected cultures, viral propagation was significantly impaired, demonstrating that the increased growth of A7-deficient strains is primarily due to enhanced cell-free propagation and virion release .

At the molecular level, A7 likely interacts with specific cellular receptors that facilitate membrane fusion between infected and uninfected cells, similar to the function of its EBV ortholog gp42, which binds to HLA class II molecules to mediate B-cell fusion . The absence of A7 may alter the viral glycoprotein complex composition on the virion surface, shifting the balance toward increased budding and release of infectious particles rather than direct cell-to-cell spread.

How does the differential effect of A7 on viral entry into different cell types (BT versus EBL cells) contribute to viral pathogenesis?

The differential effect of A7 on viral entry into different cell types reveals a sophisticated mechanism of cell tropism regulation that likely contributes to AlHV-1 pathogenesis:

• In EBL cells, A7 is required for effective viral entry, as demonstrated by significantly reduced entry efficiency of A7-deficient viruses. This suggests A7 may recognize specific receptors on these cells that are essential for viral attachment and penetration .

• Conversely, in BT cells, the absence of A7 actually improves viral entry, indicating that A7 may interfere with certain entry pathways in these cells .

This cell type-dependent effect of A7 suggests the virus may have evolved mechanisms to modulate its tropism for different tissues during infection. During natural infection, this differential tropism might allow for:

• Initial infection and replication in respiratory epithelial cells (represented by BT cells in vitro)

• Subsequent dissemination to other cell types, including the eventual targeting of CD8+ T lymphocytes

The ability to regulate entry into different cell types likely contributes to the pathogenesis of MCF by allowing the virus to establish infection in multiple tissues before reaching its main target cells (CD8+ T cells) . The absence of A7 disrupts this regulated spread, potentially explaining why A7-deficient strains fail to induce MCF despite being able to infect the host .

What interactions occur between A7 and A8 proteins during viral infection, and how do these interactions affect pathogenesis?

While the direct physical interactions between A7 and A8 proteins have not been explicitly characterized in the available search results, functional studies suggest these proteins work in concert to regulate viral spread through complementary mechanisms :

A7 and A8 appear to regulate different aspects of viral propagation:

• A7 primarily mediates cell-to-cell spread

• A8 is necessary for cell-free viral propagation

The absence of either protein renders AlHV-1 unable to induce MCF in susceptible hosts, suggesting both functions are essential for pathogenesis . This indicates that efficient viral dissemination in vivo likely requires both mechanisms of spread - cell-to-cell transmission and cell-free virion release - working in coordination.

As positional orthologs of EBV gp42 and gp350 respectively, A7 and A8 may form part of the viral entry complex on the virion surface . In EBV, gp42 and gp350 work together during viral entry, with gp350 mediating initial attachment to B cells via CD21, and gp42 triggering fusion through interaction with HLA class II molecules. A similar cooperative model may exist for AlHV-1, where A8 mediates initial attachment to target cells, while A7 facilitates subsequent fusion steps in a cell type-specific manner .

What are the optimal methods for generating recombinant AlHV-1 with A7 mutations?

The generation of recombinant AlHV-1 with A7 mutations can be accomplished through bacmid-based mutagenesis technology. Based on the methodologies described in the research literature, the following protocol represents an optimal approach :

• BAC-based mutagenesis system:

  • Start with a bacterial artificial chromosome (BAC) containing the complete AlHV-1 C500 genome

  • Use two-step galactokinase (galK) positive/negative selection in E. coli to introduce specific mutations in the A7 gene

• Specific mutation strategies for A7:

  • For complete knockout: Introduction of a stop codon early in the A7 coding sequence

  • Research has successfully employed two different A7STOP constructs:

    • A7STOP-39: Stop codon at amino acid position 39

    • A7STOP-207: Stop codon at position 207

• Reconstitution of infectious virus:

  • Transfect the modified BAC into permissive cells (bovine turbinate cells)

  • Excise the BAC backbone using Cre recombinase to restore the native viral genome structure

  • Harvest and verify the recombinant virus through sequencing

• Verification of mutant phenotype:

  • Confirm loss of A7 expression through immunoblotting or immunofluorescence

  • Perform growth kinetics and plaque formation assays in different cell types (BT and EBL)

  • Verify altered spread patterns using cell-free and cell-associated propagation assays

This methodology allows for precise genetic manipulation while preserving the remainder of the viral genome, enabling specific attribution of phenotypic changes to the targeted A7 mutations.

What assays effectively distinguish between cell-to-cell spread and cell-free propagation of AlHV-1?

Several complementary assays can effectively distinguish between cell-to-cell spread and cell-free propagation of AlHV-1 :

What animal models are appropriate for studying A7's role in MCF pathogenesis?

The rabbit model has been established as an appropriate and reliable experimental system for studying A7's role in MCF pathogenesis :

• Rabbit model advantages:

  • Rabbits are susceptible to AlHV-1 and develop clinical signs similar to MCF in cattle

  • The disease progression is more rapid than in natural hosts (typically 3-6 weeks post-infection)

  • This model allows for controlled experimental conditions and reproducible outcomes

  • Researchers have successfully used rabbits to demonstrate that both A7 and A8 are essential for MCF induction

• Experimental protocol:

  • Administer intravenous injection of defined doses of wild-type or recombinant AlHV-1

  • Monitor rabbits daily for clinical signs of MCF, including:

    • Hyperthermia (fever >40°C)

    • Weight loss

    • Depression

    • Ocular/nasal discharge

  • Euthanize animals upon development of clinical signs or at predetermined endpoints

  • Perform comprehensive post-mortem examination and tissue collection

• Key measurements and analyses:

  • Clinical scoring of MCF symptoms

  • Serological testing for anti-AlHV-1 antibodies

  • Quantification of viral genome copies in peripheral blood mononuclear cells (PBMCs), lymph nodes, and spleen using qPCR

  • Assessment of CD8+ T cell expansion and activation through flow cytometry

  • Histopathological examination of tissues

• Findings from rabbit model studies:

  • Wild-type C500 strain induces typical MCF symptoms and CD8+ T cell expansion

  • A7STOP mutants fail to induce MCF despite evidence of infection (seroconversion and detection of viral genomes in the spleen)

  • Similarly, A8STOP mutants and the attenuated WC11 strain do not cause MCF

  • Viral genomes are detectable at significantly lower levels in rabbits infected with mutant viruses compared to wild-type

The rabbit model has thus proven invaluable in demonstrating that A7 is essential for MCF pathogenesis, likely through its role in facilitating viral spread to reach CD8+ T lymphocytes, the primary cellular targets in MCF.

How does the absence of A7 affect the ability of AlHV-1 to induce MCF in susceptible hosts?

The absence of A7 completely abolishes the ability of AlHV-1 to induce malignant catarrhal fever in susceptible hosts, despite the virus retaining the ability to establish infection :

In experimental rabbit infection studies, animals infected with A7STOP mutants:

• Did not develop any clinical signs of MCF

• Did not exhibit the characteristic expansion of infected CD8+ T cells

• Showed evidence of infection (seroconversion) but at significantly reduced levels

• Had detectable viral genomes in the spleen, albeit at much lower levels than wild-type virus

• Displayed no detectable virus in peripheral blood mononuclear cells (PBMCs) or lymph nodes

This dramatic attenuation demonstrates that A7 is essential for the pathogenesis of MCF. The most likely explanation for this phenomenon is that A7 is required for efficient viral spread in vivo, particularly for reaching and establishing infection in CD8+ T lymphocytes, which are the primary cellular targets in MCF pathogenesis .

The evidence suggests that in the absence of A7, AlHV-1 can establish initial infection but cannot efficiently disseminate to reach its target cells, resulting in a subclinical infection rather than the fatal lymphoproliferative disease typically observed with wild-type virus .

What is the relationship between A7-mediated viral spread mechanisms and CD8+ T cell infection in MCF?

The relationship between A7-mediated viral spread mechanisms and CD8+ T cell infection in MCF represents a critical aspect of AlHV-1 pathogenesis :

• Role of A7 in reaching CD8+ T cells:

  • A7 appears to be essential for AlHV-1 to efficiently reach and infect CD8+ T lymphocytes in vivo

  • In rabbits infected with A7STOP mutants, there is no expansion of infected CD8+ T cells, unlike with wild-type virus

  • This suggests A7-mediated cell-to-cell spread may be particularly important for transmission of the virus to CD8+ T cells from initially infected cells

• Potential mechanisms:

  • A7 may facilitate direct cell-to-cell spread from infected antigen-presenting cells to CD8+ T cells during immune synapse formation

  • As a C-type lectin-like protein, A7 might interact with specific receptors on CD8+ T cells

  • The cell-to-cell spread mechanism promoted by A7 may help the virus evade neutralizing antibodies during dissemination to T cells

  • The differential effects of A7 on viral entry into different cell types suggest it may specifically enhance entry into T lymphocytes

• Implications for MCF pathogenesis:

  • MCF is characterized by uncontrolled proliferation and activation of latently infected CD8+ T cells

  • The inability of A7-deficient viruses to cause MCF correlates with their failure to establish infection in CD8+ T cells

  • This indicates that the specific targeting of CD8+ T cells, mediated at least in part by A7, is a prerequisite for MCF development

The data collectively suggest a model where A7 facilitates viral dissemination to CD8+ T cells through promoting cell-to-cell spread, potentially during immunological synapse formation between infected antigen-presenting cells and T lymphocytes. Once established in CD8+ T cells, the virus can then induce the lymphoproliferative pathology characteristic of MCF .

What are the potential applications of A7 research for vaccine development against MCF?

Research on the A7 protein of AlHV-1 offers promising avenues for vaccine development against malignant catarrhal fever, with several strategic approaches emerging from current findings :

• Attenuated live vaccines:

  • A7-deficient viruses (such as A7STOP mutants) represent potential live-attenuated vaccine candidates

  • These viruses can establish infection without causing disease, potentially inducing protective immunity

  • The research demonstrates that A7STOP mutants are significantly attenuated yet can still infect the host, as evidenced by seroconversion

  • Such viruses might elicit immune responses against multiple viral antigens while avoiding the development of MCF

• Subunit vaccines:

  • Understanding A7's role in pathogenesis supports the development of subunit vaccines that include key immunogenic proteins but exclude virulence factors

  • A vaccine formulation might include viral envelope proteins other than A7, potentially inducing neutralizing antibodies that block initial infection

  • This approach avoids the risk of reversion to virulence associated with attenuated live vaccines

• Vectored vaccines:

  • A7 research provides insights into essential antigens that could be expressed in heterologous viral vectors

  • Such vectors could express multiple AlHV-1 antigens except A7 and A8, potentially inducing protection without pathogenesis

  • This approach combines safety with the ability to induce broad immune responses

• Rational vaccine design principles derived from A7 research:

  • Target immune responses to viral entry and spread mechanisms

  • Block the initial infection of cell types that serve as conduits for viral spread to CD8+ T cells

  • Induce neutralizing antibodies against viral glycoproteins involved in cell entry

  • Prime T cell responses against viral antigens presented early in infection

The demonstration that both A7 and A8 are essential for MCF pathogenesis but serve different functions in viral spread suggests that targeting both proteins simultaneously might provide more robust protection. Additionally, the identification of these proteins as key virulence factors enables the development of DIVA (Differentiating Infected from Vaccinated Animals) vaccines, which would be valuable for disease control programs in regions where MCF is endemic .

What remaining questions about A7 require further investigation?

Despite significant advances in understanding the role of A7 in AlHV-1 biology and pathogenesis, several critical questions remain that warrant further investigation :

• Molecular structure and interaction partners:

  • What is the three-dimensional structure of the A7 protein?

  • What cellular receptors does A7 interact with during viral entry and cell-to-cell spread?

  • Does A7 form complexes with other viral glycoproteins, particularly A8?

  • How does the C-type lectin domain of A7 contribute to its function?

• Cell type-specific effects:

  • What is the molecular basis for A7's differential effects on viral entry into different cell types?

  • Which specific cell types in vivo require A7 for efficient infection?

  • What is the exact mechanism by which A7 mediates the infection of CD8+ T cells?

  • Are there host factors that interact with A7 in a cell type-specific manner?

• Functional domains:

  • Which domains of A7 are essential for cell-to-cell spread versus inhibiting cell-free propagation?

  • Can these functions be separated through targeted mutations?

  • How do post-translational modifications affect A7 function?

• In vivo dynamics:

  • What is the exact pathway of viral dissemination from the initial infection site to CD8+ T cells?

  • At what stage of infection does A7 play its most critical role?

  • How does the immune response target A7, and does this selection pressure drive viral evolution?

  • Can A7-deficient viruses establish latent infection, and if so, in which cell types?

• Therapeutic targets:

  • Can antibodies or small molecules targeting A7 prevent or treat MCF?

  • Would combination approaches targeting both A7 and A8 be more effective?

  • Could inhibitors of A7-receptor interactions be developed as antiviral therapeutics?

• Evolutionary considerations:

  • How conserved is A7 across different strains of AlHV-1 and related malignant catarrhal fever viruses?

  • What selective pressures have shaped A7 evolution in natural reservoir hosts versus susceptible species?

  • Do natural genetic variations in A7 correlate with differences in virulence or host range?