Recombinant Bat coronavirus Rp3/2004 Envelope small membrane protein (E)

What is the Recombinant Bat coronavirus Rp3/2004 Envelope small membrane protein (E)?

The Recombinant Bat coronavirus Rp3/2004 Envelope small membrane protein (E) is a 76-amino acid viral protein isolated from a SARS-like coronavirus (SL-CoV) found in Chinese horseshoe bats (Rhinolophus species). It belongs to the Bat coronavirus Rp3/2004 strain, which shows significant genetic similarity to the human SARS coronavirus. The E protein is a structural protein that plays crucial roles in viral assembly, budding, and pathogenesis .

The recombinant form is typically produced in expression systems such as E. coli or baculovirus and can be tagged for purification purposes. This protein has the UniProt ID Q3I5J3 and is encoded by the E gene (also known as sM), corresponding to ORF4 in the viral genome .

What are the optimal storage conditions for Recombinant Bat coronavirus Rp3/2004 E protein?

For optimal stability and activity, the Recombinant Bat coronavirus Rp3/2004 E protein should be stored according to these guidelines:

• Store at -20°C for short-term storage

• For extended storage, maintain at -80°C

• Avoid repeated freeze-thaw cycles which can compromise protein integrity

• For working stocks, store aliquots at 4°C for up to one week

• The protein is typically stored in Tris-based buffer with 50% glycerol to maintain stability

When reconstituting lyophilized protein, use deionized sterile water to a concentration of 0.1-1.0 mg/mL and add glycerol (final concentration of 6-50%) for long-term storage. Brief centrifugation of the vial prior to opening is recommended to bring contents to the bottom .

How can researchers optimize expression of Recombinant Bat coronavirus Rp3/2004 E protein?

Based on established protocols for coronavirus envelope proteins, successful expression of the Recombinant Bat coronavirus Rp3/2004 E protein requires careful optimization:

• Expression System Selection: Both prokaryotic (E. coli) and eukaryotic (baculovirus-insect cell) systems have been used successfully. The baculovirus expression system often yields proteins with proper folding and post-translational modifications.

• Vector Design Considerations:

  • Include appropriate tags (His-tag, GST-tag) for purification

  • Optimize codon usage for the host expression system

  • Include a signal sequence if secretion is desired

• Expression Conditions:

  • For E. coli: Induce at OD600 of 0.6-0.8 with IPTG concentrations of 0.1-1.0 mM

  • For insect cells: Harvest 48-72 hours post-infection

  • Lower induction temperatures (16-25°C) may improve solubility

• Purification Strategy:

  • Affinity chromatography based on the fusion tag

  • Size exclusion chromatography for final polishing

  • Consider detergent inclusion for membrane protein solubilization

How can Virus-Like Particles (VLPs) be generated using Bat coronavirus Rp3/2004 E protein?

Generation of Virus-Like Particles (VLPs) incorporating the Bat coronavirus Rp3/2004 E protein involves co-expression with other structural proteins. The established methodology includes:

• Generation of Recombinant Baculoviruses:

  • Create a baculovirus expressing the S protein of Bat coronavirus Rp3/2004 (vAcBS)

  • Create a second baculovirus expressing the E and M proteins (can be from SARS-CoV or Bat-CoV)

• Co-infection Protocol:

  • Infect insect cells (typically Sf9 or Hi5) with both recombinant baculoviruses

  • Optimal MOI (multiplicity of infection) ratio should be determined experimentally

  • Incubate at 27°C for 72-96 hours

• VLP Purification:

  • Harvest supernatant and/or cell lysate

  • Purify through sucrose cushion ultracentrifugation (typically 20-60% gradient)

  • Further purify by size exclusion chromatography

• Verification Methods:

  • Electron microscopy to confirm VLP formation

  • Western blot to verify protein incorporation

  • Immunogold labeling to confirm S protein incorporation into VLPs

This protocol allows for the generation of BVLPs (Bat coronavirus Virus-Like Particles) that can be used for immunological studies, vaccine development, and investigation of viral assembly mechanisms .

What immunological effects have been observed with VLPs containing Bat coronavirus Rp3/2004 E protein?

VLPs containing Bat coronavirus Rp3/2004 E protein (BVLPs) have demonstrated significant immunomodulatory effects in experimental studies:

• Dendritic Cell Activation:

  • Upregulation of co-stimulatory molecules: CD40, CD80, CD86, and CD83

  • Enhanced secretion of cytokines including IL-6, IL-10, and TNF-α

• Comparative Immunostimulatory Potency:

  • BVLPs showed 2-6 fold higher induction of IL-6 and TNF-α compared to SARS-CoV VLPs

  • This stronger immunostimulatory capacity is attributed primarily to differences in the S protein

• T Cell Response:

  • Increased populations of IFN-γ+ and IL-4+ CD4+ T cells when co-cultured with dendritic cells pre-exposed to BVLPs

  • Enhanced T cell responses compared to SARS-CoV VLPs

• Antibody Response:

  • SL-CoV S DNA vaccine evoked more vigorous antibody responses than SARS-CoV S DNA in mice

These findings suggest that the Bat coronavirus Rp3/2004 structural proteins, including the E protein in combination with other viral proteins, have distinct immunomodulatory properties that may inform vaccine development strategies and understanding of pathogenesis mechanisms .

How does Bat coronavirus Rp3/2004 relate to SARS-CoV and other bat coronaviruses?

Bat coronavirus Rp3/2004 belongs to the group of SARS-related coronaviruses (SARSr-CoV) found in horseshoe bats (Rhinolophus species) and shows significant genetic relationship to human SARS-CoV:

• Sequence Homology:

  • BtCoV/Rp3/2004 is 78.0% identical and 86.8% similar to SARS-CoV in the whole Spike protein

  • The E protein shows high conservation among SARS-related coronaviruses

  • Molecular clock analysis indicates that SARSr-CoVs emerged around 1972, with divergence between civet and bat strains occurring around 1995

• Comparative Analysis with Other Bat Coronaviruses:

  • Among SARS-related bat coronaviruses, Rp3/2004 clusters with strains from Chinese horseshoe bats

  • It shows distinct evolutionary characteristics compared to European bat coronavirus strains like BtCoV/BM48-31/Bulgaria/2008

• Receptor Binding Domain:

  • Critical spike domains at positions 472 and 487, important for host receptor binding, show conservation between Bat-CoV and SARS-CoV

This evolutionary relationship provides important insights into the origin of SARS-CoV and the potential for future cross-species transmission events .

What recombination events have been identified involving Bat coronavirus Rp3/2004?

Genetic analysis has revealed significant recombination events involving Bat coronavirus Rp3/2004:

• Recombination Between Bat Coronavirus Strains:

  • Evidence of frequent recombination between different SARSr-Rh-BatCoV strains

  • Recombination detected between SARSr-Rh-BatCoV Rp3 from Guangxi, China, and Rf1 from Hubei, China

• Role in SARS-CoV Evolution:

  • Civet SARSr-CoV SZ3 appears to be a recombinant virus arising from SARSr-CoV strains closely related to SARSr-Rh-BatCoV Rp3 and Rf1

  • The recombination breakpoint has been identified at the nsp16/spike region

• Ecological Factors Facilitating Recombination:

  • Migration patterns of horseshoe bats (1.86 to 17 km) allow mixing of viral strains from different geographical locations

  • This foraging range enables viral recombination between strains of different origins

These recombination events, coupled with rapid evolution particularly in the ORF7b/ORF8 region, may have contributed to the cross-species transmission and emergence of SARS-CoV .

What methods are recommended for detecting and quantifying Bat coronavirus Rp3/2004 in experimental samples?

For detection and quantification of Bat coronavirus Rp3/2004 in experimental samples, researchers commonly employ the following methodologies:

• RT-PCR Based Detection:

  • Conventional RT-PCR using primers targeting conserved regions

  • Quantitative real-time RT-PCR for viral load determination

  • Nested PCR approaches for increased sensitivity

• Primer Design Considerations:

  • Target conserved regions in the RdRp gene for broad coronavirus detection

  • Specific primers for E gene can provide selective detection of Rp3/2004

  • Typical PCR conditions: 40 cycles of 94°C (1 min), 48°C (1 min), 72°C (1 min)

• Serological Detection:

  • ELISA using recombinant E protein as capture antigen

  • Western blot analysis for protein expression confirmation

  • Immunofluorescence assays for cell culture systems

• Electron Microscopy:

  • Negative staining for visualization of viral particles

  • Immunogold labeling for specific identification of viral proteins

These techniques provide complementary approaches for comprehensive detection and characterization of Bat coronavirus Rp3/2004 in research samples .

How can researchers distinguish between acute and chronic infections in bat coronavirus models?

Based on ecological studies of SARS-related bat coronaviruses, distinguishing between acute and chronic infections involves several methodological approaches:

• Longitudinal Sampling:

  • Serial sampling of tagged bats over time (2 weeks to 4 months)

  • Monitoring of viral load in alimentary specimens

  • Assessment of seroconversion and antibody titers

• Clinical Parameters:

  • Body weight monitoring (SARSr-Rh-BatCoV positive bats showed lower body weights)

  • General health assessment of infected animals

  • Comparison with baseline parameters and control animals

• Virological Markers:

  • Viral RNA persistence in fecal samples (viral clearance typically occurs between 2 weeks and 4 months)

  • Correlation between neutralizing antibody presence and viral load

  • Assessment of subgenomic RNA as marker of active replication

• Statistical Analysis:

  • Time-to-event analysis for determining infection duration

  • Correlation analysis between antibody titers and viral loads

  • Multivariate analysis to identify factors associated with viral persistence

These approaches have established that SARSr-Rh-BatCoV causes acute, self-limiting infections in horseshoe bats, with most animals clearing the virus within several months .

How can Bat coronavirus Rp3/2004 E protein contribute to coronavirus vaccine development?

The Bat coronavirus Rp3/2004 E protein offers several valuable applications in coronavirus vaccine development:

• VLP-Based Vaccine Platforms:

  • Co-expression of E with S and M proteins creates immunogenic VLPs

  • These VLPs can serve as non-infectious vaccine candidates

  • Evidence suggests stronger immune responses compared to SARS-CoV counterparts

• Immunological Advantages:

  • BVLPs containing Rp3/2004 components showed enhanced dendritic cell activation

  • Greater induction of pro-inflammatory cytokines (2-6 fold higher production of IL-6 and TNF-α)

  • More robust T cell responses as measured by IFN-γ and IL-4 expression

• DNA Vaccine Approaches:

  • SL-CoV S DNA vaccines have demonstrated superior antibody responses

  • Inclusion of E protein genes may enhance vaccine efficacy

  • Potential for multivalent vaccine constructs targeting multiple coronavirus proteins

• Safety Considerations:

  • Modified E proteins with reduced ion channel activity may decrease pathogenicity

  • Deletion or mutation of specific E protein domains can create attenuated vaccine candidates

  • Understanding E protein's role in immune modulation helps balance immunogenicity and safety

These applications highlight the potential of Bat coronavirus Rp3/2004 E protein in developing next-generation coronavirus vaccines with improved efficacy and safety profiles .

What bioinformatic approaches are recommended for analyzing E protein conservation and function?

For comprehensive analysis of Bat coronavirus Rp3/2004 E protein conservation and function, researchers should employ these bioinformatic approaches:

• Sequence Analysis Tools:

  • Multiple sequence alignment (MUSCLE, CLUSTAL) to identify conserved domains

  • BLAST searches against coronavirus databases to determine homology

  • Protein family (Pfam) analysis to identify functional domains

• Structural Prediction Methods:

  • Transmembrane domain prediction (TMHMM, Phobius)

  • Protein secondary structure prediction (PSIPRED, JPred)

  • Homology modeling based on known coronavirus E protein structures

  • Molecular dynamics simulations to predict conformational stability

• Evolutionary Analysis:

  • Phylogenetic tree construction (Maximum Likelihood, Bayesian methods)

  • Selection pressure analysis (dN/dS ratio calculation)

  • RdRp-based grouping units (RGU) analysis with 4.8% amino acid distance threshold for alphacoronaviruses and 6.3% for betacoronaviruses

  • Recombination detection (RDP, SimPlot)

• Functional Prediction:

  • Protein-protein interaction prediction

  • Ion channel functionality analysis

  • Prediction of post-translational modifications

  • Epitope mapping for immunogenicity assessment

These computational approaches provide valuable insights into E protein evolution, structure-function relationships, and potential targets for therapeutic intervention .