Recombinant Equine arteritis virus Glycoprotein 4 (GP4)
Description
Overview of GP4
GP4 is a minor structural glycoprotein of EAV, derived from the viral open reading frame 4 (ORF4) . GP4 is a class I integral membrane protein with a molecular mass of approximately 28 kDa . It possesses three functional N-glycosylation sites, with little of its carboxy terminus exposed .
Synthesis and Processing
GP4's synthesis and processing have been characterized through in vitro translation of RNA transcripts and expression in cell culture .
Key observations include:
-
In vitro Translation: In a rabbit reticulocyte lysate system, GP4 is initially translated as a protein of about 15 kDa in the absence of microsomal membranes . In the presence of microsomal membranes, this size increases to approximately 28 kDa, suggesting glycosylation .
-
Expression in Cells: When ORF4 is expressed in baby hamster kidney cells using a vaccinia virus expression system, GP4 is synthesized and glycosylated .
-
Glycosylation Analysis: Treatment with PNGase F reduces the molecular mass of GP4 from 28 kDa to approximately 13 kDa, confirming the presence of three N-linked glycosylation sites .
Intracellular Localization
GP4 primarily localizes to the endoplasmic reticulum (ER) in both independently expressed and EAV-infected cells . This localization was determined through biochemical analysis of oligosaccharide side chains and direct visualization using immunofluorescence studies .
Disulfide-Linked Complexes
GP4 participates in the formation of disulfide-linked complexes with other minor envelope glycoproteins . Specifically, GP4 forms heterodimers with GP2b and heterotrimers with GP2b and GP3 .
-
Heterodimer Formation: Shortly after release from infected cells, virions primarily contain cystine-linked GP2b/GP4 heterodimers .
-
Heterotrimer Formation: These heterodimers are subsequently converted into disulfide-bonded GP2b/GP3/GP4 trimers through the covalent recruitment of GP3 .
Biological Activity
The biological activity of recombinant GP4 has been determined by its binding ability in functional ELISA (enzyme-linked immunosorbent assay) . This suggests that GP4 plays a role in the virus's interaction with host cells .
Recombinant GP4
Recombinant GP4 is produced in E. coli and is available with a His-tag or Tag-free . It has a purity of >90%, as determined by SDS-PAGE, and can be used in ELISA, Western blot (WB), and immunoprecipitation (IP) assays .
Immunological Significance
The study of EAV nucleocapsid (N) protein, particularly the region within the first 69 amino acid residues, has shown potential in differentiating between pre- and post-infection serum samples . Recombinant N fusion proteins containing the first 69 (rN1-69) and 28 (rN1-28) residues can detect changes in the immune response following vaccination, even without a detectable virus-neutralizing response .
Data Table
| Feature | Description |
|---|---|
| Name | Glycoprotein 4 (GP4) |
| Source Virus | Equine Arteritis Virus (EAV) |
| Type | Class I integral membrane glycoprotein |
| Molecular Mass | ~28 kDa (glycosylated), ~15 kDa (unglycosylated), ~13 kDa (deglycosylated) |
| Glycosylation Sites | Three N-glycosylation sites |
| Location | Endoplasmic Reticulum (ER) |
| Complex Formation | Heterodimers with GP2b; Heterotrimers with GP2b and GP3 |
| Biological Activity | Binds in functional ELISA |
| Recombinant Production | Expressed in E. coli, available with His-tag or Tag-free |
| Purity | >90% by SDS-PAGE |
| Applications | ELISA, WB, IP |
| Sequence (22-152 aa) | TFYPCHAAEARNFTYISHGLGHVHGHEGCRNFINVTHSAFLYLNPTTPTAPAITHCLLLV LAAKMEHPNATIWLQLQPFGYHVAGDVIVNLEEDKRHPYFKLLRAPALPLGFVAIVYVLL RLVRWAQRCYL |
| Uniprot ID | P28994 |
| Accession Number | NP_065658.1 |
| Background | Minor envelope protein; part of the GP2b-GP3-GP4 heterotrimer, which is probably responsible for attachment to target host cells; induces virion internalization predominantly through clathrin-dependent endocytosis |
Product Specs
Note: While we prioritize shipping the format currently in stock, please specify your format preference in order notes for fulfillment.
Note: Standard shipping includes blue ice packs. Dry ice shipping requires advance notification and incurs additional charges.
The tag type is determined during production. If you require a specific tag, please inform us, and we will prioritize its development.
Target Background
KEGG: vg:921343
Q&A
What is the structural and functional role of GP4 in EAV pathogenesis?
GP4 is a class I integral membrane protein with three N-glycosylation sites, forming a heterotrimeric complex with GP2 and GP3 that mediates receptor binding and membrane fusion . To confirm its role:
-
Knockout mutagenesis: Inactivation of GP4 via ATG > ACG mutations in ORF4 abolishes viral infectivity, demonstrating its indispensability .
-
Co-localization assays: GP4 co-localizes with ER and cis-Golgi markers when expressed with GP2/GP3, suggesting trafficking dependencies .
-
Disulfide linkage analysis: Non-reducing SDS-PAGE reveals covalent GP2-GP4 dimers that transition to GP2/GP3/GP4 trimers post-budding, confirmed by immunoprecipitation .
How is GP4 detected and quantified in recombinant systems?
GP4’s low abundance in virions necessitates sensitive detection methods:
-
Western blot: Use anti-tag antibodies (e.g., anti-V5 for pGP4-linker-V5 constructs) under non-reducing conditions to preserve oligomers .
-
Immunofluorescence: Transient expression in BHK-21 cells with ER (calnexin) and Golgi (GM130) markers validates subcellular localization .
-
Mass spectrometry: LC-MS/MS identifies GP4-specific peptides in purified virions, differentiating it from host proteins .
How can researchers resolve contradictions in GP4 tagging efficiency across expression platforms?
Studies report inconsistent results when tagging GP4 (e.g., failed FLAG tagging vs. successful V5 tagging) . Methodological solutions include:
-
Linker optimization: Inserting a GMAPGRDPPVAT linker between GP4 and the V5 tag improves folding and epitope accessibility .
-
Co-expression systems: Simultaneous expression of GP2/GP3/GP4 in mammalian cells (e.g., MultiMam system) enhances solubility and tag visibility .
-
Validation controls: Compare tagged GP4 functionality to wild-type using infectivity assays (e.g., TCID50) and oligomerization profiles .
What strategies address GP4’s folding challenges in prokaryotic vs. eukaryotic systems?
GP4 expressed in E. coli forms inclusion bodies, while mammalian systems yield soluble but ER-retained protein :
Refolding protocol for E. coli-derived GP4:
-
Extract inclusion bodies with 8M urea.
-
Dialyze against 20mM Tris (pH 7.4) + 150mM NaCl + 1mM GSH/GSSG.
-
Co-dialyze His-GP2b with GST-GP3/GP4 to stabilize soluble trimers .
How do GP4 interactions with GP2, GP3, and E-protein influence virion assembly?
GP4’s interplay with other structural proteins is critical for virion morphogenesis:
-
Co-immunoprecipitation: Anti-HA pulldowns in GP3-HA-tagged EAV confirm GP2/GP3/GP4 trimerization and weaker E-protein associations .
-
Trafficking blockade: Brefeldin A treatment traps GP4 in the ER, preventing Golgi-mediated oligomerization and virion incorporation .
-
Functional knockout: E-protein deletion reduces GP4 incorporation by >90%, suggesting a chaperone-like role for E in spike assembly .
What emerging techniques are advancing GP4 structural biology?
-
Cryo-ET: Resolve GP4’s spatial arrangement within the native virion spike at near-atomic resolution.
-
Alanine scanning mutagenesis: Identify GP4 residues critical for GP2/GP3 binding using yeast two-hybrid screens.
-
Single-molecule FRET: Characterize real-time conformational changes in GP4 during pH-dependent membrane fusion.
How can conflicting data on GP4’s ER retention versus Golgi trafficking be reconciled?
Discrepancies arise from expression context:
