Recombinant African swine fever virus Protein MGF 110-9L (Ken-019)

What is MGF 110-9L (Ken-019) and what is its role in African swine fever virus pathogenesis?

MGF 110-9L is a full-length protein (290 amino acids) encoded by the multigene family 110 of African swine fever virus (ASFV). The specific isolate Ken-019 refers to a Kenyan isolate of the virus. This protein belongs to a family of ASFV proteins that are critical for viral virulence and immune evasion strategies . Research has demonstrated that MGF 110-9L plays a significant role in modulating host immune responses, particularly in interfering with the type I interferon pathway, which is essential for antiviral defense mechanisms.

MGF 110-9L contributes to ASFV pathogenicity by helping the virus evade host immune detection and response. When this gene is deleted in combination with other virulence factors like MGF505-7R (creating ASFV-Δ110-9L/505-7R), the resulting virus demonstrates attenuated virulence in pigs while maintaining immunogenicity, making it a promising candidate for vaccine development .

How is recombinant MGF 110-9L (Ken-019) protein typically expressed and purified for research applications?

Recombinant MGF 110-9L (Ken-019) protein is commonly expressed in E. coli expression systems with an N-terminal His-tag to facilitate purification . The standard protocol involves:

• Cloning the full-length (1-290aa) coding sequence into a suitable expression vector with an N-terminal His-tag

• Transformation into E. coli expression strains

• Induction of protein expression under optimized conditions

• Cell lysis and extraction of the protein

• Purification via immobilized metal affinity chromatography (IMAC)

• Further purification steps as needed to achieve >90% purity as verified by SDS-PAGE

• Lyophilization of the purified protein for stable storage

For reconstitution of the lyophilized protein, it's recommended to:

• Briefly centrifuge the vial to collect contents

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add 5-50% glycerol (with 50% being optimal) for long-term storage

• Aliquot and store at -20°C/-80°C to avoid repeated freeze-thaw cycles

What experimental approaches are used to study MGF 110-9L function in ASFV pathogenesis?

Multiple experimental approaches are employed to study MGF 110-9L function in ASFV pathogenesis:

• Gene deletion studies: Creating recombinant viruses with MGF 110-9L deletions (alone or in combination with other genes) to assess changes in virulence and pathogenicity. The ASFV-Δ110-9L/505-7R construct is an example of this approach .

• Protein-protein interaction assays: Techniques such as co-immunoprecipitation, yeast two-hybrid, and proximity ligation assays to identify host cellular proteins that interact with MGF 110-9L.

• Transcriptomic and proteomic analyses: RNA sequencing (RNA-seq) and Western blotting to identify changes in host gene expression and protein levels in response to MGF 110-9L. For example, RNA-seq revealed that ASFV-Δ110-9L/505-7R weakened TBK1 degradation compared to wild-type ASFV .

• Immunological assays: Measurement of cytokine production, particularly type I interferons, in response to infection with wild-type versus MGF 110-9L-deleted viruses.

• Cellular localization studies: Immunofluorescence microscopy to determine the subcellular localization of MGF 110-9L during infection and its co-localization with host factors.

• In vivo challenge studies: Infection of pigs with wild-type versus attenuated viruses lacking MGF 110-9L to assess clinical outcomes, immune responses, and protection against challenge with virulent strains .

How does MGF 110-9L interact with host immune response pathways?

MGF 110-9L significantly impacts host immune response pathways through several mechanisms:

• Autophagy modulation: Research has demonstrated that MGF 110-9L, in conjunction with other viral proteins, influences autophagy pathways. When MGF 110-9L is deleted (as in ASFV-Δ110-9L/505-7R), TBK1 degradation through the autophagy pathway is inhibited .

• Type I interferon suppression: Wild-type ASFV containing MGF 110-9L suppresses type I interferon responses. The deletion mutant ASFV-Δ110-9L/505-7R shows reduced ability to suppress this key antiviral pathway, leading to enhanced interferon production .

• PIK3C2B interaction: Studies have identified that the autophagy-related protein PIK3C2B is involved in the inhibition of TBK1 degradation induced by ASFV-Δ110-9L/505-7R. This suggests that MGF 110-9L normally interferes with PIK3C2B function to promote TBK1 degradation and suppress immune responses .

• Innate immune evasion: MGF 110-9L likely contributes to ASFV's ability to evade innate immune detection and response, enabling the virus to establish infection and replicate effectively in host cells before adaptive immunity can be mobilized.

What are the optimal conditions for working with recombinant MGF 110-9L in laboratory settings?

For optimal handling and use of recombinant MGF 110-9L protein in laboratory settings:

Storage and Stability:

• Store lyophilized protein at -20°C/-80°C upon receipt

• After reconstitution, store working aliquots at 4°C for up to one week

• For long-term storage, add glycerol to a final concentration of 50% and store aliquots at -20°C/-80°C

• Avoid repeated freeze-thaw cycles as this degrades the protein

Reconstitution Protocol:

• Centrifuge the vial briefly before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to 0.1-1.0 mg/mL

• The protein is typically stored in Tris/PBS-based buffer with 6% Trehalose at pH 8.0

Experimental Applications:

• For SDS-PAGE applications, the protein shows greater than 90% purity

• When designing binding or functional assays, consider that the His-tag may influence protein folding or interactions

• For cell-based assays, determine optimal concentrations through titration experiments

Quality Control Measures:

• Verify protein integrity by SDS-PAGE before use

• For functional studies, include appropriate positive and negative controls to validate activity

• Consider the potential influence of the His-tag on structure and function when interpreting results

What is the mechanism by which MGF 110-9L deletion contributes to virus attenuation?

The deletion of MGF 110-9L, particularly in combination with MGF505-7R (ASFV-Δ110-9L/505-7R), contributes to virus attenuation through several intricate mechanisms:

• Enhanced TBK1 stability: Research has demonstrated that ASFV-Δ110-9L/505-7R weakens TBK1 degradation compared to wild-type ASFV. TBK1 is a critical kinase in the type I interferon production pathway, and its preservation leads to stronger antiviral responses that limit viral replication .

• Altered autophagy dynamics: MGF 110-9L deletion disrupts the virus's ability to manipulate host autophagy pathways. Specifically, ASFV-Δ110-9L/505-7R blocked the degradation of TBK1 through the autophagy pathway, suggesting that MGF 110-9L normally facilitates TBK1 targeting for autophagic degradation .

• PIK3C2B regulation: The autophagy-related protein PIK3C2B is downregulated in ASFV-Δ110-9L/505-7R infection, and this downregulation is involved in inhibiting TBK1 degradation. This indicates that MGF 110-9L may normally interact with or regulate PIK3C2B to promote autophagy-mediated immune suppression .

• Enhanced interferon response: The preservation of TBK1 in ASFV-Δ110-9L/505-7R infection leads to increased type I interferon production, creating a stronger antiviral state that restricts viral replication and spread, thereby attenuating virulence while maintaining immunogenicity .

This attenuation mechanism represents a promising approach for developing live attenuated vaccines against ASFV, as it reduces virulence while preserving protective immunogenicity.

How does MGF 110-9L affect TBK1 degradation and autophagy pathways?

MGF 110-9L plays a sophisticated role in modulating TBK1 degradation and autophagy pathways:

• Promotion of TBK1 degradation: Wild-type ASFV containing MGF 110-9L promotes the degradation of TBK1, a key kinase in the interferon production pathway. When MGF 110-9L is deleted (as in ASFV-Δ110-9L/505-7R), TBK1 degradation is impaired, as confirmed by both RNA-seq and Western blotting analyses .

• Autophagy-mediated degradation: Research has confirmed that ASFV-Δ110-9L/505-7R blocked the degradation of TBK1 specifically through the autophagy pathway. This suggests that MGF 110-9L normally functions to target TBK1 for selective autophagy, resulting in its degradation and subsequent suppression of interferon responses .

• PIK3C2B interactions: Studies have identified that the autophagy-related protein PIK3C2B is involved in this process. The downregulation of PIK3C2B was observed in ASFV-Δ110-9L/505-7R infection and was associated with the inhibition of TBK1 degradation. This suggests that MGF 110-9L may normally upregulate or interact with PIK3C2B to promote autophagy-mediated TBK1 degradation .

• Viral replication impact: Interestingly, PIK3C2B was found to promote ASFV-Δ110-9L/505-7R replication in vitro, indicating a complex relationship between autophagy modulation and viral replication efficiency .

These findings reveal a sophisticated mechanism by which MGF 110-9L contributes to ASFV immune evasion by manipulating cellular autophagy pathways to degrade key components of antiviral signaling.

What are the promising experimental approaches for developing MGF 110-9L-based vaccines?

Several promising experimental approaches have emerged for developing MGF 110-9L-based vaccines:

• Gene deletion combinations: Combining MGF 110-9L deletion with other virulence gene deletions has shown significant promise. The ASFV-Δ110-9L/505-7R construct demonstrated complete protection against parental lethal ASFV challenge, indicating effective immunogenicity despite attenuation .

• Attenuated live virus vaccines: Table of attenuated ASFV candidates and their protective efficacy:

• Mechanism-based attenuation: Understanding the role of MGF 110-9L in TBK1 degradation and type I interferon suppression provides a rational basis for vaccine development. By specifically targeting these immune evasion functions, more precise attenuation strategies can be developed .

• Combination with immune modulators: Since MGF 110-9L normally suppresses type I interferon responses, combining MGF 110-9L deletion with adjuvants or immune modulators that further enhance interferon responses could potentially improve vaccine efficacy.

• Recombinant subunit vaccines: Using recombinant MGF 110-9L protein as part of a subunit vaccine formulation, possibly combined with other immunogenic ASFV proteins, represents another approach worth investigating.

The most promising approach currently appears to be gene-deleted live attenuated vaccines, as multiple studies have demonstrated their ability to provide protective immunity against challenge with virulent ASFV strains .

How do mutations or variants in MGF 110-9L affect ASFV virulence across different isolates?

The impact of MGF 110-9L mutations or variants on ASFV virulence varies across different viral isolates:

• Sequence variation: Analysis of MGF 110-9L across ASFV isolates reveals both conserved and variable regions. The Ken-019 isolate (P0C9J0) contains the full 290 amino acid sequence, but other isolates may contain variations that affect protein function and virulence .

• Functional conservation: Despite sequence variations, the immune evasion function of MGF 110-9L appears to be conserved across multiple ASFV genotypes. Deletion of this gene consistently contributes to attenuation in various strain backgrounds, suggesting a fundamental role in virulence .

• Strain-specific effects: The magnitude of attenuation achieved by MGF 110-9L deletion varies between ASFV strains. This suggests that the relative contribution of MGF 110-9L to virulence may differ depending on the genetic background of the virus and potentially the presence of compensatory mechanisms in some isolates.

• Combinatorial effects: The most effective attenuation strategies involve deleting MGF 110-9L in combination with other virulence genes. The specific combination that yields optimal attenuation while preserving immunogenicity may vary between ASFV strains:

• In the CN/GS/2018 strain, combining MGF 110-9L and MGF505-7R deletions produced an effective attenuated vaccine candidate

• Other effective combinations have included multiple members of the MGF110 family with genes from other multigene families

• Host adaptation: The function of MGF 110-9L may also be influenced by host adaptation. ASFV isolates that have adapted to different host or vector species may show variations in MGF 110-9L function and contribution to virulence.

Understanding these variations is critical for developing broadly effective vaccine strategies that can protect against diverse ASFV isolates.