Recombinant African swine fever virus Protein MGF 360-3L (Mal-016)

What is Recombinant ASFV Protein MGF 360-3L (Mal-016)?

Recombinant ASFV Protein MGF 360-3L (Mal-016) is a viral protein derived from African swine fever virus, specifically from the isolate Tick/Malawi/Lil 20-1/1983. It belongs to the multigene family 360 (MGF360) of ASFV proteins, which are generally involved in host immune evasion mechanisms. The recombinant protein is commonly produced in expression systems such as Escherichia coli, and in commercial preparations, it typically encompasses amino acids 1-286 of the native viral protein . The protein is identified in protein databases with Uniprot ID: 0C9J5 and constitutes part of the virus's arsenal of immunomodulatory factors that interfere with host defense mechanisms.

Where is MGF 360-3L located in the ASFV genome?

MGF 360-3L is part of the multigene family 360 (MGF360) located in the variable regions of the ASFV genome. Based on genomic organization patterns of ASFV, most MGF360 genes are located in the left terminal repeat region of the genome, similar to other family members such as MGF360-1L through MGF360-15L . The genomic positioning of these genes in the terminal variable regions suggests their potential role in adaptation and host-range determination. The specific nucleotide positions vary across different ASFV isolates, but the gene typically maintains its relative position and order within the genome architecture of diverse ASFV strains.

How conserved is MGF 360-3L across different ASFV isolates?

MGF 360-3L shows significant conservation across various ASFV isolates, although specific sequence variations occur based on geographical origin and evolutionary history. Most MGF360 family proteins contain a characteristic Pfam ASFV360 domain that identifies them as members of this family . The conservation status of MGF360 proteins suggests their functional importance in viral replication and host interaction processes. While some MGF360 genes can be partially deleted during stable cell adaptation leading to decreased virulence in swine, the core functional domains often remain conserved across virulent field isolates, indicating their potential significance in natural infection cycles.

What is the role of MGF 360-3L in ASFV pathogenesis?

While the specific role of MGF 360-3L has not been fully characterized, insights can be derived from studies of related MGF360 family proteins. Members of the MGF360 family are known to participate in host immune evasion strategies, particularly in modulating interferon responses. For instance, MGF360-4L has been demonstrated to negatively regulate the cGAS-STING mediated type I interferon signaling pathway by inhibiting interferon-β promoter activity and reducing transcriptional levels of interferon-stimulated genes . MGF360-4L accomplishes this by interacting with IRF3 and suppressing its phosphorylation, thereby inhibiting a key transcription factor in antiviral responses . The deletion of multiple MGF360 genes can result in attenuated virulence, suggesting that these proteins collectively contribute to ASFV pathogenicity by undermining host immune defenses.

How does MGF 360-3L interact with host cellular components?

Research on related MGF360 family proteins provides a framework for understanding potential MGF 360-3L interactions. For example, MGF360-16R specifically interacts with SERTA domain-containing 3 (SERTAD3) and syndecan-binding protein (SDCBP), suggesting a role in modulating host cell transcriptional activity . Similarly, MGF360-4L co-localizes and interacts with IRF3, inhibiting its phosphorylation and activation . Based on functional similarities within the MGF360 family, MGF 360-3L likely targets specific host proteins involved in innate immune signaling pathways. Potential interaction partners could include components of the interferon induction or signaling pathways, pattern recognition receptors, or transcriptional regulators that control antiviral gene expression.

What molecular mechanisms does MGF 360-3L employ to evade host immune responses?

Drawing from studies of MGF360 family proteins, MGF 360-3L likely employs several molecular mechanisms to subvert host immunity. Research on MGF360-4L has shown that it inhibits the cGAS-STING signaling pathway at multiple levels: it suppresses interferon-β promoter activity induced by cGAS/STING, TBK1, and IRF3-5D; reduces mRNA levels of interferon-β and interferon-stimulated genes; and disrupts antiviral responses, leading to increased susceptibility to other viral infections . The protein accomplishes this by interacting with IRF3 and inhibiting its phosphorylation, thereby blocking its activation and nuclear translocation . MGF 360-3L may utilize similar mechanisms, potentially targeting different components of interferon signaling pathways or employing distinct strategies to achieve immune evasion.

What expression systems are optimal for producing recombinant MGF 360-3L?

The optimal expression system for recombinant MGF 360-3L production is Escherichia coli, which has been successfully used for the expression of various ASFV proteins, including the commercially available recombinant MGF 360-3L protein . For E. coli expression, the gene encoding MGF 360-3L should be codon-optimized for bacterial expression and cloned into an appropriate expression vector containing a strong inducible promoter (e.g., T7) and affinity tags for purification. Alternative expression systems include insect cells (using baculovirus vectors) for proteins requiring eukaryotic post-translational modifications, or mammalian expression systems for functional studies requiring proper folding and modification in a context more closely resembling the natural host environment.

What cell-based assays can effectively measure MGF 360-3L's impact on interferon signaling?

Several cell-based assays can effectively measure the impact of MGF 360-3L on interferon signaling, based on methodologies used with related ASFV proteins. Dual-luciferase reporter assays using IFN-β promoter-driven luciferase constructs provide a sensitive readout of signaling pathway activation . In this approach, cells (such as HEK293T) are co-transfected with IFN-β-luc, pRL-TK (for normalization), pathway activators (e.g., cGAS/STING, TBK1, or IRF3-5D), and MGF 360-3L expression plasmids. After 24 hours, promoter activity is measured using dual-luciferase reporter assay kits .

Additionally, quantitative PCR can assess the effect of MGF 360-3L on mRNA levels of interferon-β and interferon-stimulated genes (ISGs) like ISG15, ISG54, and ISG56 . Western blotting using phospho-specific antibodies can detect the impact on phosphorylation of signaling components like IRF3 and TBK1 . Viral challenge assays using interferon-sensitive viruses like VSV-GFP can evaluate the functional consequence of MGF 360-3L expression on antiviral responses .

What techniques are most effective for analyzing MGF 360-3L protein-protein interactions?

Based on research with related MGF360 family proteins, several techniques are effective for analyzing protein-protein interactions involving MGF 360-3L. Co-immunoprecipitation (co-IP) is a foundational approach, where cells are transfected with tagged versions of MGF 360-3L and potential interaction partners, followed by immunoprecipitation with antibodies against one protein and western blotting to detect the co-precipitated partner . Confocal microscopy using fluorescently tagged proteins can visualize co-localization, providing spatial information about interactions .

Additional advanced techniques include proximity ligation assays for detecting interactions in situ, and FRET (Förster Resonance Energy Transfer) or BRET (Bioluminescence Resonance Energy Transfer) for analyzing interactions in live cells. For unbiased discovery of interaction partners, mass spectrometry-based approaches following immunoprecipitation of MGF 360-3L from infected cells or expression systems can identify novel binding proteins. Yeast two-hybrid screening provides another method for identifying potential interactors from cDNA libraries.

How do the functional domains of MGF 360-3L compare with other MGF360 family members?

Analysis of MGF360 family members reveals important functional domains that may be conserved in MGF 360-3L. For MGF360-4L, studies have identified critical immunosuppressive domains through truncation mutant analysis. Specifically, the regions 4L-F2 (75-162 aa) and 4L-F3 (146-387 aa) were found to contain the crucial immunosuppressive domains responsible for inhibiting interferon signaling . The table below summarizes the identified functional domains and their activities:

MGF 360-3L likely contains similar functional domains given the conservation patterns within the MGF360 family, but specific mapping would require similar truncation analysis. The Pfam ASFV360 domain (typically spanning approximately 200 amino acids) is a characteristic feature that defines the MGF360 family and is likely present in MGF 360-3L, potentially mediating its immunomodulatory functions .

What are the effects of MGF 360-3L deletion on ASFV virulence and replication?

In contrast, deletion of six MGF genes (three from MGF360 and three from MGF505) resulted in complete attenuation in swine while still conferring protection against homologous wild-type challenge . This suggests that while individual MGF360 genes may be dispensable, collective deletion of multiple family members significantly impacts virulence. The specific contribution of MGF 360-3L would need to be determined through targeted gene deletion studies, assessing both in vitro replication in macrophage cultures and in vivo virulence in appropriate animal models.

How can CRISPR/Cas9 technology be applied to study MGF 360-3L function?

CRISPR/Cas9 technology offers powerful approaches for studying MGF 360-3L function in various contexts. For viral genome editing, CRISPR/Cas9 can be used to precisely delete, mutate, or tag MGF 360-3L within the ASFV genome. This approach allows for the generation of recombinant viruses with specific modifications to MGF 360-3L while maintaining the integrity of the rest of the genome. The methodology involves designing guide RNAs targeting sequences flanking the MGF 360-3L gene, along with a repair template containing desired modifications.

For cellular studies, CRISPR/Cas9 can be employed to knockout host proteins that potentially interact with MGF 360-3L, thereby elucidating their roles in MGF 360-3L-mediated immune evasion. Additionally, CRISPR activation (CRISPRa) or CRISPR interference (CRISPRi) systems can be used to modulate the expression of host factors without completely eliminating them, providing insights into dose-dependent effects. CRISPR screening approaches can identify host factors required for MGF 360-3L function by conducting genome-wide or targeted screens in relevant cell types.

What are the key research gaps in understanding MGF 360-3L function?

Several critical research gaps exist in understanding MGF 360-3L function. First, the specific host targets and molecular mechanisms of MGF 360-3L remain largely uncharacterized, unlike some other MGF360 family members. Second, the structural features that mediate these interactions have not been determined. Third, the relative contribution of MGF 360-3L to ASFV virulence compared to other immune evasion proteins requires systematic analysis through combinatorial gene deletion studies. Fourth, the potential for MGF 360-3L-based interventions, such as attenuated vaccines or targeted antivirals, remains unexplored. Finally, the evolutionary conservation and variation of MGF 360-3L across ASFV isolates need comprehensive analysis to understand the selective pressures shaping this protein.

How might MGF 360-3L contribute to ASFV vaccine development?

MGF 360-3L presents several potential applications in ASFV vaccine development. Understanding its role in immune evasion could inform rational attenuation strategies. If MGF 360-3L significantly contributes to virulence while being nonessential for replication, its deletion could create live-attenuated vaccine candidates that maintain immunogenicity while reducing pathogenicity. Research on MGF360-4L suggests it is a potential candidate gene for ASFV live-attenuated vaccines due to its role in immune evasion . Similar approaches could be applied to MGF 360-3L if it demonstrates comparable functions.

Additionally, recombinant MGF 360-3L protein could serve as a subunit vaccine component or as a marker for differentiating infected from vaccinated animals (DIVA) in marker vaccine strategies. Comprehensive understanding of the immunomodulatory mechanisms employed by MGF 360-3L and other MGF360 family proteins is essential for designing vaccines that effectively overcome ASFV-mediated immune suppression and induce protective immunity.