Recombinant Orgyia pseudotsugata multicapsid polyhedrosis virus Occlusion-derived virus envelope protein E56 (ODVP6E)

What is the structural characterization of ODVP6E protein?

ODVP6E (ODV-E56) is a structural component of occlusion-derived viruses (ODVs) that is primarily associated with the viral envelope. Research indicates that this protein can be detected as a 15 kDa polypeptide in both the envelope and nucleocapsid fractions of virions, though it appears to be enriched in the envelope fraction . The protein is encoded by specific open reading frames in different baculoviruses, including ORF56 in Bombyx mori nucleopolyhedrovirus . Full-length ODVP6E from Orgyia pseudotsugata multicapsid polyhedrosis virus consists of 374 amino acids and is commonly expressed with histidine tags in recombinant systems .

Methodologically, researchers can fractionate virions into envelope and nucleocapsid components using detergent treatments followed by western blotting with specific antibodies to determine the localization of ODVP6E within the viral structure. This approach has been validated through comparison with other viral proteins such as VP39 (capsid), PIF1 (ODV envelope), and GP64 (BV envelope) .

How does ODVP6E differ across baculovirus species?

ODVP6E exhibits structural conservation across different baculovirus species but with notable sequence variations. Comparative analysis of the recombinant full-length proteins shows:

These subtle differences in length may contribute to host-specific adaptations while maintaining the core functional domains required for viral infection . To investigate these variations, researchers typically employ sequence alignment tools and structural prediction algorithms to identify conserved domains and species-specific regions that might influence host range or infection efficiency.

What is the temporal expression pattern of ODVP6E during viral infection?

ODVP6E expression follows the temporal regulation pattern typical of baculovirus structural proteins. While the search results don't specifically detail ODVP6E expression timing, comparable envelope proteins follow patterns where their expression is regulated during the infection cycle. In studies of the Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus infection cycle, structural proteins like p39 are detected on Western blots at specific time points post-infection .

The temporal expression pattern can be experimentally determined through time-course analyses using Western blotting or quantitative PCR at various time points post-infection (p.i.). For instance, early viral genes like gp64 begin expression shortly after infection, while structural proteins associated with the capsid or envelope typically follow later in the infection cycle . To properly characterize ODVP6E expression, researchers should collect samples at multiple time points (e.g., 6, 12, 24, 48, and 72 hours p.i.) and analyze protein levels using antibodies specific to ODVP6E.

How does ODVP6E contribute to the ODV entry complex and host cell infection?

ODVP6E functions as a component of the ODV entry complex, which is critical for primary infection of the host midgut epithelium. Similar to other per os infectivity factors (PIFs), ODVP6E likely participates in host cell recognition and viral entry mechanisms. Research on related baculovirus envelope proteins suggests that these proteins form a stable complex that mediates ODV entry into host cells .

The ODV entry complex consists of a stable core (including proteins like PIF1-4) and several loosely associated components. Like Ac108, ODVP6E may be essential for the formation of the complete ODV entry complex, as studies have shown that the absence of certain components prevents complex formation . The methodological approach to investigating ODVP6E's role would include:

• Generation of ODVP6E-knockout viruses using CRISPR-Cas9 or traditional recombination techniques

• Analysis of complex formation using blue native PAGE and Western blotting

• Oral infectivity assays comparing wild-type and ODVP6E-deficient viruses

• Co-immunoprecipitation studies to identify protein-protein interactions within the entry complex

These experimental approaches would help determine whether ODVP6E is essential for complex integrity and functional viral entry.

What are the optimal conditions for expressing and purifying recombinant ODVP6E protein?

Expressing and purifying recombinant ODVP6E requires optimization of several parameters to ensure high yield and biological activity. Based on established protocols for baculovirus envelope proteins:

Bacterial Expression System:

• E. coli is the predominant expression system for recombinant ODVP6E

• BL21(DE3) or Rosetta strains are recommended for expressing viral envelope proteins

• Induction conditions: 0.5-1.0 mM IPTG at 16-25°C for 16-20 hours to minimize inclusion body formation

• Addition of 0.1-0.5% Triton X-100 to lysis buffer helps solubilize membrane-associated proteins

Purification Protocol:

• For His-tagged ODVP6E: Ni-NTA affinity chromatography with imidazole gradient elution (50-250 mM)

• Size exclusion chromatography to remove aggregates and ensure monodispersity

• Ion exchange chromatography as a polishing step

• Concentrate to 1-5 mg/mL in a buffer containing 25 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol

Quality control should include SDS-PAGE, Western blotting, and mass spectrometry to confirm protein identity and purity. Additionally, circular dichroism spectroscopy can assess proper protein folding before functional assays.

How does multiplicity of infection (MOI) affect ODVP6E expression and function?

Multiplicity of infection (MOI) significantly influences the infection dynamics of baculoviruses and consequently the expression of structural proteins like ODVP6E. Research on Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus has shown that MOI affects the magnitude but not the timing of early events in viral infection .

When designing experiments to optimize ODVP6E expression:

• Higher MOI (e.g., 100) increases the percentage of initially infected cells, potentially leading to earlier and more synchronous ODVP6E expression

• Lower MOI (e.g., 5-10) may result in multiple rounds of infection and potentially higher final yields

• The timing of DNA synthesis (12-18 hours post-infection) and budded virus production (peaking at 24-36 hours post-infection) are relatively consistent across different MOI values

For functional studies, researchers should consider that MOI influences:

• The timing of viral gene expression

• The final rates of budded virus production

• The relative timing of structural protein appearance

Therefore, experimental designs should account for these factors when studying ODVP6E expression and function, with MOI selection based on whether synchronous infection or maximum yield is the primary goal.

What techniques are most effective for studying ODVP6E localization within infected cells?

Studying ODVP6E localization requires sophisticated imaging techniques combined with appropriate sample preparation. The most effective approaches include:

• Immunofluorescence microscopy:

  • Fix cells at various time points post-infection with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Incubate with anti-ODVP6E primary antibodies and fluorophore-conjugated secondary antibodies

  • Co-stain with markers for cellular compartments (ER, Golgi, nucleus)

  • Analyze using confocal microscopy for precise subcellular localization

• Immunoelectron microscopy:

  • Process infected cells for transmission electron microscopy

  • Perform immunogold labeling with anti-ODVP6E antibodies

  • This approach provides nanometer-scale resolution of ODVP6E localization within virions and cellular structures

• Subcellular fractionation:

  • Separate nuclear, cytoplasmic, and membrane fractions from infected cells

  • Analyze fractions by Western blotting to track ODVP6E distribution over time

  • Compare with markers for different cellular compartments

• Live-cell imaging:

  • Generate recombinant viruses expressing ODVP6E fused to fluorescent proteins (e.g., GFP)

  • Monitor protein trafficking in real-time using spinning disk confocal microscopy

  • Correlate with viral morphogenesis stages

These techniques should be employed at multiple time points post-infection to capture the dynamic localization of ODVP6E during the viral replication cycle.

How can researchers effectively identify protein-protein interactions involving ODVP6E?

Understanding the protein interaction network of ODVP6E is crucial for elucidating its functional roles. Several complementary techniques can be employed:

• Co-immunoprecipitation (Co-IP):

  • Prepare lysates from infected cells or purified virions

  • Use anti-ODVP6E antibodies coupled to protein A/G beads

  • Analyze precipitated proteins by mass spectrometry to identify interaction partners

  • Validate identified interactions with reverse Co-IP and Western blotting

• Proximity-based labeling:

  • Generate BioID or TurboID fusions with ODVP6E

  • Express in insect cells during viral infection

  • Identify biotinylated proteins using streptavidin pulldown and mass spectrometry

  • This approach can capture transient interactions and proteins in close proximity

• Blue native PAGE:

  • This technique has been successfully used to analyze the ODV entry complex

  • Solubilize viral envelopes under native conditions

  • Separate protein complexes by electrophoresis

  • Identify components by Western blotting or mass spectrometry

• Yeast two-hybrid screening:

  • Use ODVP6E as bait against a library of baculovirus or host proteins

  • Validate positive interactions with targeted assays

  • This approach can identify direct binary interactions

• Crosslinking mass spectrometry:

  • Treat purified virions or infected cells with protein crosslinkers

  • Digest and analyze by mass spectrometry

  • Identify crosslinked peptides to determine interaction interfaces

These methods collectively provide a comprehensive view of ODVP6E's interaction landscape, revealing its role in complex formation and viral processes.

What are the best approaches for analyzing the function of ODVP6E in viral entry?

Analyzing ODVP6E's function in viral entry requires a combination of genetic, biochemical, and cell biological approaches:

• Generation of knockout and mutant viruses:

  • Create ODVP6E-deficient viruses using CRISPR-Cas9 or homologous recombination

  • Generate point mutations in conserved domains to identify functional residues

  • Complement with wild-type or mutant ODVP6E to confirm specificity

• Oral infectivity assays:

  • Compare wild-type and ODVP6E-mutant viruses in per os infections of susceptible insects

  • Determine median lethal dose (LD50) and time to death

  • Quantify viral replication in midgut epithelial cells

• Cell binding and entry assays:

  • Label purified ODVs with fluorescent dyes

  • Measure binding to isolated midgut cells or cultured insect cells

  • Track internalization using confocal microscopy and flow cytometry

  • Compare wild-type and ODVP6E-deficient viruses

• Liposome binding studies:

  • Prepare liposomes mimicking the composition of insect midgut membranes

  • Assess binding of purified ODVP6E using flotation assays or surface plasmon resonance

  • Determine lipid specificity and binding kinetics

• Structure-function analysis:

  • Express truncated or domain-specific ODVP6E variants

  • Assess their ability to integrate into membranes and interact with other viral proteins

  • Perform competitive inhibition assays with peptides derived from ODVP6E

These methodologies provide complementary information about ODVP6E's role in viral entry, from whole organism pathogenesis to molecular interactions.

How can ODVP6E be utilized in baculovirus-based gene delivery systems?

ODVP6E has potential applications in enhancing baculovirus-based gene delivery systems. Baculoviruses have been widely used as gene expression tools and are being developed for gene therapy applications . Incorporating knowledge about ODVP6E could improve these systems through:

• Enhanced viral entry:

  • Engineering modified ODVP6E to increase viral tropism for specific mammalian cell types

  • Creating fusion proteins combining ODVP6E with cell-targeting ligands or antibodies

  • Optimizing the ratio of envelope proteins to enhance transduction efficiency

• Display platforms:

  • Using ODVP6E as a scaffold for displaying heterologous proteins on the viral surface

  • Engineering ODVP6E to present antigens for vaccine development

  • Creating baculovirus display libraries for protein-protein interaction studies

• Targeted gene delivery:

  • Modifying ODVP6E similar to how GP64 has been engineered to enhance transduction

  • Incorporating cell-specific targeting motifs into ODVP6E

  • Developing hybrid envelope systems combining ODVP6E with components from other viruses

• Improving transduction efficiency:

  • Combining ODVP6E modifications with other approaches like the expression of VP39 fused with protein transduction domains

  • Optimizing the stoichiometry of envelope proteins to enhance cellular uptake

  • Engineering ODVP6E to resist inactivation by complement or other host defense factors

These approaches require detailed understanding of ODVP6E structure-function relationships and its interactions with both viral and cellular components.

What are the key challenges in studying post-translational modifications of ODVP6E?

Investigating post-translational modifications (PTMs) of ODVP6E presents several methodological challenges:

• Identification challenges:

  • Insect cell-specific modifications may differ from those in bacterial expression systems

  • Low abundance of specific modified forms requires enrichment techniques

  • Comprehensive PTM mapping requires multiple complementary analytical approaches

• Methodological approaches:

  • Mass spectrometry-based proteomics with multiple fragmentation techniques (CID, ETD, HCD)

  • Enrichment strategies for specific modifications (phosphopeptides, glycopeptides)

  • Site-directed mutagenesis of putative modification sites

  • Comparison of recombinant protein from bacterial and insect expression systems

• Functional significance determination:

  • Generation of viruses with mutations at modification sites

  • Comparative structural studies of modified and unmodified forms

  • In vitro assays to assess the impact of modifications on protein-protein interactions

  • Cell-based assays to evaluate effects on viral entry and infectivity

• Temporal dynamics:

  • Tracking changes in modifications throughout the viral replication cycle

  • Correlating modification patterns with localization and function

  • Identifying the enzymes responsible for adding and removing modifications

These challenges necessitate an integrated approach combining advanced proteomics, molecular biology, and functional assays to fully characterize ODVP6E post-translational modifications.

How does host specificity influence ODVP6E function across different insect species?

ODVP6E likely plays a role in determining host range specificity of baculoviruses. Understanding this relationship requires comparative studies across different viral and host species:

• Comparative genomics approach:

  • Analyze sequence conservation and variation in ODVP6E across baculovirus species

  • Correlate sequence features with known host ranges

  • Identify potential host-interacting domains through evolutionary analysis

• Host range determination methods:

  • Cross-infection studies using recombinant viruses with chimeric ODVP6E proteins

  • In vitro binding assays with midgut cells from various insect species

  • Competition assays with species-specific ODVP6E variants

• Molecular determinants of specificity:

  • Identify specific amino acid residues or structural features that confer host specificity

  • Create chimeric proteins swapping domains between ODVP6E from different viral species

  • Perform structural analysis of ODVP6E interaction with host receptors

• Experimental validation approach:

  • Generate recombinant baculoviruses expressing ODVP6E from different viral sources

  • Test infection efficiency and pathogenesis in various host species

  • Quantify binding affinity to midgut cells or membranes from different insects

This research would contribute to understanding the molecular basis of baculovirus host range and potentially enable the development of more targeted biological control agents or gene delivery systems with modified host specificity.