Recombinant Lactate dehydrogenase-elevating virus Protein X (VPX)

What is Lactate Dehydrogenase-Elevating Virus (LDV) and why is it important for research?

LDV is an enveloped arterivirus composed of a single strand of RNA that naturally infects mice. It belongs to the Arteriviridae family, related to coronaviruses such as SARS . The virus is named for its characteristic elevation of lactate dehydrogenase levels in infected mice due to impairment of enzyme clearance mechanisms .

LDV is important for research for several reasons:

• It establishes persistent infections regardless of mouse strain, age, sex, or immune status

• It causes rapid but transient activation of both B cells and T cells in lymphoid tissues

• It serves as a model for studying innate immune responses, particularly Type I interferon production

• It provides insights into mechanisms of viral persistence despite robust immune responses

• It has unique interactions with specific macrophage populations that can inform broader understanding of virus-host cell dynamics

What are the structural and functional characteristics of LDV Protein X (VPX)?

While the search results don't provide specific information about a protein named "VPX" in LDV, viral proteins in arteriviruses typically serve multiple functions. Based on general virology principles and the available information about LDV, viral proteins would likely be involved in:

• Viral replication and transcription within macrophages

• Modulation of host immune responses, particularly interferon pathways

• Structural components of the viral particle

• Mediation of host cell entry and viral assembly

Structural analysis would require protein expression and purification, followed by techniques such as X-ray crystallography or cryo-electron microscopy to determine three-dimensional structure. Functional analysis would involve creating recombinant proteins, performing binding assays, and conducting mutation studies to identify key functional domains.

What experimental systems are available for studying recombinant LDV proteins?

Researchers studying recombinant viral proteins typically utilize several experimental systems:

• Bacterial expression systems: E. coli-based expression for high yields of protein, though often lacking post-translational modifications

• Mammalian cell culture: HEK293 or similar cell lines for expression with appropriate mammalian post-translational modifications

• Insect cell/baculovirus systems: Intermediate between bacterial and mammalian systems, offering good yields with some post-translational modifications

• Cell-free expression systems: For proteins that might be toxic to cell-based systems

For functional studies, researchers can use:

• Primary mouse macrophage cultures (LDV's natural target)

• Mouse models, particularly focusing on TLR7 knockout mice which have been shown to have altered responses to LDV infection

• In vitro binding and activity assays using purified recombinant proteins

How can I verify the purity and identity of recombinant LDV VPX protein?

Verification of recombinant viral protein identity and purity involves multiple analytical approaches:

• SDS-PAGE: To assess protein size and initial purity

• Western blotting: Using specific antibodies, if available, to confirm protein identity

• Mass spectrometry: For precise molecular weight determination and peptide mapping

  • MALDI-TOF or LC-MS/MS can be used to confirm amino acid sequence

• Circular dichroism: To assess secondary structure content

• Dynamic light scattering: To evaluate protein homogeneity and detect aggregation

• Size exclusion chromatography: To analyze oligomeric state and purity

For proteomics approaches, proper experimental design is crucial. As noted in search result , parameters such as sample preparation, protein separation, MS detection limit, and MS dynamic range all significantly influence experimental outcomes.

How does LDV VPX interact with the host immune system and what methodologies best capture these interactions?

LDV has been shown to induce rapid lymphocyte activation mediated by type I interferons, particularly IFNα. This process appears to be dependent on plasmacytoid dendritic cells (pDCs) and TLR7 signaling . To study potential interactions between viral proteins and immune components:

• Co-immunoprecipitation assays: To identify direct protein-protein interactions

• Surface plasmon resonance: For quantitative analysis of binding kinetics

• Proximity labeling approaches: Such as BioID or APEX to identify interaction partners in living cells

• CRISPR-Cas9 screens: To identify host factors required for viral protein function

• Flow cytometry: To assess effects on immune cell activation, using markers such as CD69

• Ex vivo cultures with neutralizing antibodies: As described in the research where anti-IFNα antibodies prevented LDV-induced CD69 upregulation

Methodological approach for immune interaction studies:

• Isolate splenic B cells using CD19+ magnetic beads

• Culture cells with plasma from LDV-infected mice (collected at appropriate timepoints)

• Add recombinant VPX protein or vehicle control

• Assess activation markers such as CD69 via flow cytometry

• Include appropriate controls such as neutralizing antibodies against IFNα

What experimental design considerations are critical for proteomics studies involving LDV VPX?

When designing proteomics experiments to study viral proteins like LDV VPX, several critical factors must be considered:

• Sample preparation:

  • Protein separation techniques (fractionation)

  • Digestion efficiency (typically using trypsin)

  • Prevention of protein/peptide loss during preparation

• Mass spectrometry parameters:

  • Detection limit

  • Dynamic range

  • Resolution

• Experimental variables:

  • Technical replicates to assess methodological variation

  • Biological replicates to capture biological variation

  • Appropriate controls

Based on the modeling described in search result , the success rate of protein detection and the relative dynamic range (RDR) are significantly influenced by:

• The degree of protein separation

• The MS detection limit

• The MS dynamic range

Importantly, the sequence in which these parameters are optimized matters. For example, improving protein separation followed by enhancing detection limit and then dynamic range produces better results than improving protein separation followed by enhancing dynamic range and then detection limit .

How can I detect significant changes in LDV VPX abundance or modifications during infection?

Detecting significant changes in viral protein abundance requires robust statistical approaches. According to search result , empirical Bayes methods that shrink a protein's sample variance toward a pooled estimate provide more powerful and stable inference compared to ordinary t-tests.

Recommended methodological approach:

• Experimental design:

  • Use balanced designs with adequate biological replicates

  • Consider isobaric mass labeling techniques (e.g., iTRAQ)

  • Include appropriate controls

• Data analysis steps:

  • Normalize mass spectrometry data to account for technical variation

  • Apply moderated test statistics using empirical Bayes methods

  • For case-control experiments, use linear mixed effects models to adjust for potential differences due to channel effects, loading, mixing, and sample handling

  • Simultaneously estimate protein relative abundance and assess differential expression

• For two-group comparisons (e.g., infected vs. uninfected):

  • Compare log2 relative abundances

  • Apply statistics that account for multiple testing

  • Use appropriate visualization methods to present results

The computational approach can be simplified by using median sweeps for protein abundance estimation, which reduces computational cost while maintaining efficiency and robustness .

What are the best approaches for studying the structural changes of LDV VPX in different chemical environments?

Understanding how viral proteins change structure in different environments is critical for elucidating their function. Search result discusses protein conformational changes in different solutions, which can be applied to studying viral proteins:

Methodological approaches:

• Fluorescence spectroscopy:

  • Intrinsic fluorescence from tryptophan and tyrosine residues

  • Extrinsic fluorescence using probes that bind to hydrophobic regions

  • Changes in fluorescence intensity indicate conformational changes, as seen when proteins are exposed to different solutions like DPC versus aqueous environments

• Circular dichroism (CD) spectroscopy:

  • Far-UV CD for secondary structure determination

  • Near-UV CD for tertiary structure insights

  • Thermal denaturation studies to assess stability

• Nuclear Magnetic Resonance (NMR) spectroscopy:

  • For detailed atomic-level structural information

  • Particularly useful for monitoring structural changes in different conditions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Maps solvent-accessible regions of proteins

  • Identifies conformational changes upon exposure to different conditions

The example from search result demonstrates how a protein's surface properties can change dramatically in different environments, with hydrophilic residues predominating on the surface in aqueous solution and hydrophobic residues becoming exposed in the presence of DPC. This principle is important for understanding how viral proteins might change conformation when interacting with different cellular compartments or membranes.

How does TLR7 signaling influence LDV infection and what techniques can assess this relationship?

LDV infection induces a strong IFNα response that is dependent on TLR7 signaling in plasmacytoid dendritic cells. Research has shown that:

• TLR7 knockout mice fail to mount IFNα responses to LDV infection

• This lack of IFNα response prevents CD69 upregulation on lymphocytes

• Despite the absence of IFNα response, viral titers are not significantly affected

Methodological approach to study TLR7-viral protein interactions:

• In vivo studies:

  • Compare TLR7 wild-type and knockout mice

  • Measure IFNα levels in plasma using ELISA

  • Assess lymphocyte activation via flow cytometry (CD69 expression)

  • Determine viral titers through quantitative PCR or plaque assays

• In vitro studies:

  • Isolate pDCs from wild-type and TLR7 knockout mice

  • Expose to recombinant viral proteins

  • Measure IFNα production

  • Use TLR7 agonists and antagonists as controls

• Molecular binding studies:

  • Express recombinant TLR7 and viral proteins

  • Perform binding assays (co-IP, SPR)

  • Use mutagenesis to identify key interaction residues

The data table below summarizes findings from a representative experiment comparing TLR7 wild-type and knockout mice:

This table demonstrates that while TLR7 signaling is critical for the interferon response and lymphocyte activation, it does not significantly impact viral replication .

What are the most reliable methods for detecting LDV infection in laboratory mice?

LDV infection can be detected through several complementary approaches:

• Lactate dehydrogenase (LDH) enzyme levels:

  • Measure LDH levels in plasma or serum

  • Elevated levels indicate possible LDV infection

  • Note: False positives can occur due to trauma to cardiac muscles during cardiocentesis, inflammation, or other stressors

• Molecular detection:

  • PCR-based detection from blood or tissue samples

  • Highly specific and recommended for confirmation

  • Can be used to screen blood or tumors from mice suspected of infection

• Immunological methods:

  • Multiplexed fluorescent immunoassay (MFI)

  • Immunofluorescence assay (IFA)

  • Detect viral antigens or antibodies against viral proteins

• Experimental inoculation:

  • Transfer suspected infected material to naive mice

  • Monitor for LDH elevation and other signs of infection

  • Useful for confirming infectious potential

For research facilities, a combined approach using LDH screening followed by PCR confirmation provides the most reliable detection protocol. The limitation of relying solely on LDH elevation is the possibility of false positives from other sources of cellular damage .

How can I distinguish between different strains of LDV using recombinant viral proteins?

While the search results don't provide specific information about distinguishing LDV strains, general virology principles suggest several approaches:

• Sequence-based differentiation:

  • PCR amplification and sequencing of strain-specific regions

  • Strain-specific PCR primers designed to target variable regions

  • Restriction fragment length polymorphism (RFLP) analysis

• Protein-based approaches:

  • Develop strain-specific antibodies against recombinant viral proteins

  • Perform Western blot analysis to detect strain-specific epitopes

  • Use ELISA with strain-specific antibodies

• Functional assays:

  • Compare neurotropic vs. non-neurotropic strains' ability to induce paralysis in susceptible mouse strains (C58 or AKR) when ecotropic mouse leukemia virus is present

  • Assess differences in replication kinetics in macrophage cultures

  • Measure interferon induction capacity between strains

A robust differentiation protocol would combine sequence analysis with functional assays to confirm strain identity, particularly when distinguishing between neurotropic and non-neurotropic variants.

What are the most promising research directions for understanding LDV VPX function?

Future research on LDV Protein X (VPX) could profitably focus on:

• Structural biology approaches:

  • Determining high-resolution structures using X-ray crystallography or cryo-EM

  • Mapping functional domains through mutagenesis studies

• Host-pathogen interaction studies:

  • Identifying cellular binding partners using proximity-based labeling approaches

  • Characterizing effects on host cell signaling pathways

• Immunomodulatory function:

  • Investigating potential role in TLR7 signaling modulation

  • Exploring impact on IFNα production and response

• Therapeutic and vaccine applications:

  • Evaluating VPX as a potential target for antivirals

  • Assessing immunogenicity and potential for vaccine development

• Evolutionary studies:

  • Comparative analysis with related arteriviruses

  • Investigation of adaptive mutations under selective pressure