Recombinant Vesicular stomatitis New Jersey virus Glycoprotein G (G)

Basic Research Questions

• What is the structure and function of VSV-NJ glycoprotein G?

  Vesicular stomatitis New Jersey virus (VSNJV) glycoprotein G (G protein) is a 517-amino acid transmembrane protein that serves as the sole surface protein responsible for viral attachment and membrane fusion . The G protein attaches to host cellular receptors, inducing clathrin-dependent endocytosis of the virion. In the endosome, the acidic pH induces conformational changes in the glycoprotein trimer, which trigger fusion between virus and endosomal membrane . The protein contains two glycosylation sites at positions 178 and 335, similar to the Indiana serotype, but differs in that it is not acylated . Structurally, G protein forms trimeric complexes that undergo significant conformational rearrangements during the fusion process . In neurons, neo-synthesized glycoproteins are sorted to the dendrites, where the virus buds .

• What methods can verify the proper folding and function of recombinant VSV-NJ glycoprotein G?

  Verifying proper folding and function involves several complementary approaches:

  • Functional ELISAs: Measuring binding ability using monoclonal antibodies specific to conformational epitopes

  • SDS-PAGE analysis: Confirming protein size and purity (>90% purity should be achieved)

  • Neutralization assays: Testing whether antibodies against the recombinant protein neutralize authentic virus

  • Flow cytometry: Analyzing cell surface expression in transfected cells using specific antibodies

  • Cell fusion assays: Evaluating the ability of the G protein to mediate pH-dependent membrane fusion

  • Incorporation into pseudotyped virus particles: Assessing whether the protein can be incorporated into virions and mediate infection

  Researchers should employ multiple methods to comprehensively validate the structural and functional integrity of recombinant G proteins before proceeding with downstream applications.

Advanced Research Questions

• How can one optimize recombinant VSV-NJ glycoprotein G for diagnostic ELISA development?

  Optimization of recombinant VSNJV G for diagnostic ELISA development involves several critical steps:

  1. Antigen extraction and purification: Extract glycoprotein from partially purified VSV-NJ to achieve optimal purity while maintaining native conformation

  2. Selection of appropriate monoclonal antibodies: Identify and validate neutralizing MAbs specific to VSV-NJ to develop a competitive blocking ELISA

  3. Cutoff determination: Establish appropriate threshold values by correlating ELISA inhibition percentages with virus neutralization test (VNT) titers; for example, 40% inhibition was found to correspond to a VNT titer of 32

  4. Cross-reactivity testing: Validate that the assay does not cross-react with related viruses, including foot-and-mouth disease virus, swine vesicular disease virus, or VSV serotype Indiana

  5. Specificity validation: Test assay performance with naïve sera from various species (cattle, pigs, horses) to ensure high specificity (≥99.6%)

  6. Comparison with existing methods: Compare the glycoprotein-based ELISA with nucleocapsid-based ELISAs and VNTs to confirm superior performance and compatibility with gold standard methods

  The optimal expression system for diagnostic applications appears to be the baculovirus system in insect cells, which has demonstrated superior performance in producing antigens that maintain conformational epitopes required for high-specificity diagnostics .

• What approaches are effective for studying pH-dependent conformational changes of VSV-NJ glycoprotein G?

  Studying the pH-dependent conformational changes of VSV-NJ glycoprotein G requires multidisciplinary approaches:

  1. Site-directed mutagenesis: Introducing mutations at key residues (e.g., D137, E139, W72, Y73, Y116, A117) to assess their impact on pH sensitivity and fusion activity

  2. Fusion loop mutation analysis: Altering the hydrophobicity of fusion loops to produce nonfusogenic mutants (W72A, Y73A, Y116A, A117F) that can be used to dissect the fusion mechanism

  3. Second-site suppressor identification: Passage of fusion-defective mutants to identify compensatory mutations (e.g., E76K) that restore fusion activity, revealing critical structural relationships

  4. Complementation studies: Using heterologous glycoproteins (e.g., from VSV-Indiana) to rescue viruses bearing lethal mutations in the New Jersey G protein

  5. Structural biology techniques: X-ray crystallography or cryo-electron microscopy to determine the structure of G protein in pre- and post-fusion states

  6. Oligomerization analysis: Assessing how mutations affect the formation of G protein trimers, which are critical for fusion activity

  By combining these approaches, researchers can systematically map the determinants of pH-dependent conformational changes and develop a comprehensive understanding of the fusion mechanism.

• How does the sequence diversity of VSV-NJ isolates impact recombinant glycoprotein functionality?

  The extensive genetic diversity within the VSV-NJ serotype has significant implications for glycoprotein functionality:

  1. Sequence variation magnitude: Up to 19.8% G gene sequence differences exist among NJ serotype isolates, leading to up to 8.5% amino acid differences between virus isolates

  2. Evolutionary constraints: Analysis of nucleotide substitutions reveals that third-position changes are distributed randomly throughout the gene (84% of substitutions), while first and second position changes are non-random, indicating functional constraints on protein structure

  3. Conserved regions: Only three short oligonucleotide stretches show complete sequence conservation, suggesting these regions are critical for essential functions

  4. Variable domains: Clusters of amino acid substitutions are present in the hydrophobic signal sequence, transmembrane domain, and cytoplasmic domain of the G protein, potentially affecting membrane insertion, trafficking, and virion assembly

  5. Antigenic implications: Differences are located throughout the G protein, including regions adjacent to defined major antibody neutralization epitopes, potentially affecting diagnostic test and vaccine development

  6. Phylogenetic structure: The VSV-NJ serotype contains at least three distinct lineages or subtypes, with all recent US and Mexican isolates belonging to subtype I

  When designing recombinant G proteins for research or diagnostic applications, researchers should carefully consider which isolate to use as the reference sequence, and potentially develop a panel of recombinant proteins representing major genetic lineages to ensure broad coverage.

• What strategies can be employed for developing recombinant VSV-based vaccines using both New Jersey and Indiana glycoproteins?

  Multiple strategies have been developed for creating recombinant VSV vaccines incorporating both serotypes:

  1. Chimeric virus approaches: Three types of recombinant viruses have been successfully generated:

     • VSIV-GI: Single copy of the glycoprotein gene from Indiana serotype

     • VSIV-GNJ: Single copy of the glycoprotein gene from New Jersey serotype

     • VSIV-GNJGI: Two copies of the glycoprotein gene, one from each serotype

  2. Immunological considerations:

     • Single-serotype recombinant viruses (VSIV-GI, VSIV-GNJ) induce serotype-specific neutralizing antibodies

     • Dual-serotype virus (VSIV-GNJGI) induces neutralizing antibodies against both serotypes, but with Indiana serotype dominance in the serological response

  3. Protection assessment:

     • Swine immunized with VSIV-GI or VSIV-GNJ were protected against homologous high-dose virus challenge

     • Pigs inoculated with VSIV-GNJGI were protected against VSIV-GI challenge but showed limited protection against highly pathogenic New Jersey field isolates

  4. Host-specific pathogenicity:

     • In mice, VSIV-GNJ and VSIV-GNJGI were attenuated compared to VSIV-GI

     • In swine (a natural host), G-NJ glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than VSIV-GI

  This research highlights the importance of testing vaccine candidates in natural hosts, as pathogenicity patterns in laboratory animals may not predict outcomes in target species. Furthermore, the bivalent vaccine approach (VSIV-GNJGI) shows promise but requires optimization to overcome serotype dominance issues.

• What methodological approaches are effective for studying the organization of VSV-NJ glycoprotein G in membrane microdomains?

  Investigating the organization of VSNJV G protein in membrane microdomains requires specialized techniques:

  1. Immunogold-labeling electron microscopy: This technique allows visualization of G protein distribution in arbitrarily chosen areas of plasma membranes, revealing organization into microdomains with diameters of 100-150 nm

  2. Transfection studies: Expression of G protein in the absence of other viral components through transfection with constructs like pCAGGS-G allows determination of whether organization into microdomains is intrinsic to G or dependent on other viral proteins

  3. Flow cytometry analysis: Quantification of surface-expressed G protein at different time points post-infection or post-transfection to track expression dynamics

  4. Comparative analysis: Parallel studies of G protein organization in infected versus transfected cells to identify similarities and differences

  5. Time-course experiments: Analysis at various time points (e.g., 4.5, 6, 8, 10, 12, or 14 h post-infection) to track dynamic changes in membrane organization

  6. Antibody labeling: Use of specific antibodies like anti-G protein antibody I1 to selectively label and track surface-expressed G protein

  These approaches have revealed that G protein organizes predominantly into membrane microdomains independently of interactions with other virion components, suggesting an intrinsic property of the protein that may be critical for virus assembly and budding.

• How do the fusion loops of VSV-NJ glycoprotein G contribute to membrane fusion, and how can they be studied?

  The fusion loops of VSV G protein are critical for membrane fusion and can be studied through several approaches:

  1. Site-directed mutagenesis: Altering the hydrophobicity of the two fusion loops within G by creating specific mutants:

     • W72A, Y73A, Y116A, and A117F, which are nonfusogenic

     • These mutations do not affect protein oligomerization or transport to the cell surface

  2. Reverse genetics: Producing authentic recombinant viral particles bearing lethal mutations in the G gene through complementation with heterologous glycoprotein

  3. Fusion assays: Determining how mutations affect the ability of G protein to mediate acid pH-triggered membrane fusion

  4. Interference studies: Analyzing how nonfusogenic G proteins interfere with wild-type G function through mixed trimer formation or inhibition of trimer function during fusion

  5. Second site suppressor identification: Passage of mutant viruses to identify compensatory mutations that restore fusion activity, such as E76K mutation that suppresses the A117F fusion block

  6. pH sensitivity analysis: Determining how mutations affect the pH threshold for fusion, as exemplified by the E76K mutation which rendered G more sensitive to acid pH-triggered fusion

  These approaches not only confirm the critical role of hydrophobic fusion loops in membrane fusion but also highlight the importance of sequence elements surrounding the hydrophobic tips in driving the fusion process, providing targets for inhibiting G-mediated fusion.

• What considerations are important when using recombinant VSV-NJ glycoprotein G as a platform for diagnostic development?

  Developing diagnostics using recombinant VSNJV G requires attention to several critical factors:

  1. Expression system selection: For diagnostic applications, the baculovirus-insect cell system has demonstrated superior performance in maintaining conformational epitopes required for specific antibody recognition

  2. Purification strategy: Optimal diagnostic performance requires high purity (>90%) as determined by SDS-PAGE analysis

  3. Assay format optimization: Blocking ELISA using glycoprotein and monoclonal antibodies has shown excellent specificity (99.6%) and compatibility with virus neutralization tests

  4. Cross-reactivity control: Thorough validation to ensure no cross-reactivity with related viruses (foot-and-mouth disease virus, swine vesicular disease virus, VSV-Indiana)

  5. Threshold determination: Careful establishment of cutoff values through correlation with gold standard methods; a 40% inhibition threshold corresponding to a VNT titer of 32 has proven effective

  6. Analytical validation: Testing against diverse panels of known positive and negative samples from multiple species (cattle, horses, pigs) to establish performance characteristics

  7. Reference sample inclusion: Incorporation of well-characterized positive and negative controls in each assay to ensure consistency and reliability

  When properly optimized, recombinant G protein-based assays can serve as useful alternatives to virus neutralization tests for detecting antibodies specific to VSV-NJ, with advantages in terms of throughput, safety, and ease of standardization.

Experimental Design and Methodology