Recombinant Hirame rhabdovirus Matrix protein (M)

What is the genetic organization of HIRRV matrix proteins?

Unlike many rhabdoviruses, HIRRV has two matrix protein genes, M1 and M2, with complete sequences of 771 and 700 nucleotides, respectively. The M1 gene encodes a protein of 227 amino acids, while the M2 gene encodes a protein of 193 amino acids . Interestingly, the M1 gene contains an additional small open reading frame (ORF) that could encode a highly basic, arginine-rich protein of 25 amino acids, similar to the secondary ORFs found in vesicular stomatitis virus (VSV) but considerably shorter . The genetic organization suggests functional specialization between these two matrix proteins in HIRRV.

How does HIRRV M protein compare to other fish rhabdoviruses?

Sequence analysis reveals that HIRRV is more closely related to infectious hematopoietic necrosis virus (IHNV) than to viral hemorrhagic septicemia virus (VHSV), though it remains distinct from both . In the central portion of the M1 proteins of HIRRV and IHNV, there is a highly conserved region (amino acids 108 to 147) where 39 of 40 (97.5%) amino acids are identical . This conservation suggests critical functional importance of this region. The putative consensus gene termination sequence for IHNV and VHSV, AGAYAG(A)7, is present in the N-M1, M1-M2, and M2-G intergenic regions of HIRRV, as are the putative transcription initiation sequences YGGCAC and AACA .

What are the key functions of rhabdovirus M proteins?

The matrix proteins of rhabdoviruses, including HIRRV, are multifunctional. They are essential for:

• Virus assembly and maturation - binding to nucleocapsids and the cytoplasmic domain of G protein to facilitate budding

• Regulation of viral RNA synthesis - mediating a switch from transcription to replication

• Modulation of host gene expression - inhibiting host cell transcription by localizing to the nucleus

• Inducing cytopathic effects - including cell rounding (in VSV) or apoptosis (in lyssaviruses)

In HIRRV specifically, the M protein is abundant in virus particles and conserved, making it useful for phylogenetic and epidemiological studies .

How can HIRRV M protein be expressed in E. coli systems?

The recombinant expression of HIRRV M protein can be achieved using the following methodology:

• Gene amplification and cloning: Amplify the M gene using specific primers designed based on the HIRRV M gene sequence. For optimal expression in E. coli, use primers containing appropriate restriction sites (e.g., BamHI and SalI) for cloning into an expression vector such as pET-32a .

• Transformation and induction: Transform the recombinant plasmid into E. coli BL21(DE3) and induce protein expression with IPTG. SDS-PAGE analysis of induced cultures reveals a distinct band of approximately 43 kDa for the fusion protein containing the ~21 kDa His/Trx/S-tag and the M protein .

• Protein purification: Purify the recombinant M protein using Ni²⁺ affinity chromatography, which yields high-purity protein suitable for further applications .

A typical expression and purification result is shown in the following table based on analysis of SDS-PAGE gels:

What verification methods confirm the identity of recombinant HIRRV M protein?

Several methodological approaches can verify the identity of purified recombinant M protein:

• Western blotting: Use anti-HIRRV polyclonal antibodies or monoclonal antibodies specific to the M protein. Research shows that monoclonal antibodies developed against recombinant M protein recognize both the recombinant protein and a ~22 kDa protein in HIRRV-infected cells .

• Mass spectrometry: MALDI-TOF analysis of immune-reactive proteins excised from polyacrylamide gels confirms the identity of the M protein. In one study, 8 mass spectrum peaks matched with the M protein of HIRRV, covering 39% of the amino acid sequence .

• Immunofluorescence assay (IFA): Monoclonal antibodies against recombinant M protein produce strong green fluorescence signals in HIRRV-infected EPC cells, confirming the specificity of the antibodies and the identity of the recombinant protein .

How does the 3D structure of HIRRV M protein relate to its self-assembly properties?

While the specific 3D structure of HIRRV M protein has not been fully characterized, insights can be drawn from structural studies of related rhabdovirus M proteins. The matrix proteins of VSV and Lagos bat virus (LBV) share a common fold despite having no identifiable sequence homology . Both structures show a stretch of residues from the N-terminus of adjacent molecules binding to a hydrophobic cavity on the protein surface, forming non-covalent linear polymers .

For HIRRV M protein research, methodological approaches to investigate self-assembly properties could include:

• X-ray crystallography or cryo-EM: These techniques could reveal whether HIRRV M proteins form similar non-covalent polymers through N-terminal interactions with hydrophobic pockets on adjacent molecules.

• Mutagenesis studies: Targeted mutations in the N-terminal region and potential hydrophobic binding pockets could identify specific residues critical for self-association.

• In vitro polymerization assays: Monitoring the aggregation of purified recombinant M protein under different conditions (pH, salt concentration, temperature) could provide insights into the factors that regulate self-assembly.

What role does HIRRV M protein play in regulating viral RNA synthesis?

Studies with other rhabdoviruses suggest the M protein plays a crucial role in shifting the balance from transcription to replication. For HIRRV, this regulatory function could be investigated using:

• Minigenome systems: Develop a reverse genetics system where the effect of M protein on viral RNA synthesis can be quantified. The system would include a reporter gene flanked by HIRRV regulatory sequences, along with plasmids expressing N, P, and L proteins, with or without M protein .

• M protein deletion or mutation studies: Compare RNA synthesis patterns between wild-type and M-deficient or M-mutant viruses to determine how specific domains affect the balance between transcription and replication.

• RNA-protein interaction studies: Employ electrophoretic mobility shift assays (EMSA) or RNA immunoprecipitation to examine whether M protein directly interacts with viral RNA or with components of the replication complex.

How do post-translational modifications affect HIRRV M protein function?

Rhabdovirus M proteins can be phosphorylated or palmitoylated, potentially affecting their functions . For HIRRV M protein, investigating these modifications would involve:

• Phosphorylation analysis: Use mass spectrometry to identify phosphorylation sites, followed by site-directed mutagenesis to create phosphomimetic (e.g., Ser/Thr to Asp/Glu) or phospho-deficient (e.g., Ser/Thr to Ala) mutants.

• Palmitoylation studies: Employ metabolic labeling with palmitic acid analogs and click chemistry to detect and quantify palmitoylation, and identify specific cysteine residues involved using site-directed mutagenesis.

• Functional assays: Compare the membrane-binding properties, self-assembly, and effects on viral replication of wild-type versus modification-deficient M proteins.

How can recombinant HIRRV M protein be used for diagnostic purposes?

The recombinant M protein offers several methodological applications for HIRRV diagnostics:

• Development of monoclonal antibodies: Recombinant M protein can be used as an immunogen to produce monoclonal antibodies with high specificity for HIRRV. These antibodies can then be employed in various diagnostic assays .

• ELISA development: The purified recombinant M protein can be used to coat microplates for the detection of anti-HIRRV antibodies in fish serum or mucus samples. This approach employs appropriate detection antibodies such as FIgM-Mab followed by alkaline phosphatase-conjugated secondary antibodies .

• Immunohistochemistry and immunofluorescence assays: Antibodies against recombinant M protein enable the detection of HIRRV in tissue sections or cell cultures, providing spatial information about viral infection .

What approaches can be used to study HIRRV M protein interactions with host factors?

Understanding M protein-host factor interactions requires systematic methodological approaches:

• Yeast two-hybrid screening: Use the M protein as bait to screen fish cDNA libraries for potential interaction partners.

• Co-immunoprecipitation coupled with mass spectrometry: Pull down M protein complexes from infected cells and identify associated proteins by mass spectrometry.

• Proximity labeling: Employ BioID or APEX2 fusion proteins to identify proteins in close proximity to the M protein in living cells.

• Surface plasmon resonance or biolayer interferometry: Measure binding kinetics between purified recombinant M protein and candidate host factors identified through the above methods.

• Functional validation: Confirm the biological relevance of identified interactions through gene knockdown or knockout approaches in susceptible fish cell lines.

How does HIRRV M protein compare structurally and functionally with other fish rhabdovirus M proteins?

Comparative analysis requires several methodological approaches:

• Sequence alignment and phylogenetic analysis: Compare the amino acid sequences of M proteins from HIRRV, IHNV, VHSV, and other fish rhabdoviruses to identify conserved motifs and evolutionary relationships.

• Domain mapping: Through truncation mutants and chimeric proteins, identify which regions of the different M proteins are responsible for specific functions (e.g., binding to nucleocapsids, membrane association).

• Cross-complementation studies: Determine whether the M protein from one fish rhabdovirus can functionally replace the M protein of another in virus assembly or regulation of RNA synthesis.

• Structural comparison: If 3D structures are available, compare the folding patterns, surface charge distributions, and hydrophobic patches that might contribute to protein-protein interactions.

What metabolomic changes are associated with HIRRV M protein expression during infection?

Recent metabolomic studies of HIRRV infection have identified significant changes in host metabolism . To specifically attribute these changes to M protein function:

• Comparative metabolomics: Compare metabolic profiles of cells infected with wild-type HIRRV versus M-deficient or M-mutant viruses.

• Targeted metabolite supplementation: Based on identified differential metabolites (such as inosine, carnosine, glutamine, glycine, and 7-methylguanine), systematically test their effects on HIRRV replication in the presence or absence of functional M protein .

• Metabolic enzyme activity assays: Measure the activities of key metabolic enzymes in cells expressing recombinant M protein alone versus control cells.

• Stable isotope labeling: Use 13C or 15N labeled metabolic precursors to track metabolic fluxes in the presence or absence of M protein expression.

This approach could reveal whether M protein-induced metabolic changes are direct (through interaction with metabolic enzymes) or indirect (through effects on host gene expression).