HAV VP4-VP2

What is HAV VP4-VP2 and how does it differ from other picornavirus proteins?

HAV VP4-VP2 refers to capsid proteins of Hepatitis A Virus that exhibit significant structural differences compared to other picornaviruses. Most notably, HAV utilizes a VP2 "domain swap" that is characteristic of insect picorna-like viruses . This structural feature places HAV evolutionarily between typical picornaviruses and insect viruses. The VP4 component in HAV is exceptionally short compared to other picornaviruses and uniquely lacks a signal for myristoylation, which is typically considered essential for membrane penetration in other picornaviruses .

What are the applications of recombinant HAV VP4-VP2 in laboratory research?

Recombinant HAV VP4-VP2 (specifically residues 55-164) can be effectively used in ELISA and Western blot assays. It provides excellent detection capability for HAV with minimal specificity problems, making it valuable for viral detection and characterization studies . The recombinant protein has a molecular weight of 44 kDa and is typically supplied in a formulation of 10 mM CBB pH 9.6, 0.1% SDS, and 50% glycerol with purity greater than 90% as determined by SDS-PAGE .

How do the structural features of HAV capsid proteins contribute to viral stability?

HAV exhibits unusually high particle stability compared to other picornaviruses. While researchers initially hypothesized that the VP2 domain swap might explain this stability, comparative analysis with cricket paralysis virus (CrPV), which shares the same domain swap but displays typical picornavirus stability, disproved this theory . Instead, the stability appears to result from tight packing in the interaction region adjacent to the icosahedral 2-fold axes. HAV achieves this complementarity through matching small residues and tyrosine side-chains nestled around the 2-fold axis . This structural arrangement likely contributes to HAV's remarkable stability while the re-wiring of VP2 may reflect fundamental differences in genome uncoating mechanisms compared to enteroviruses.

What is known about the cleavage of VP0 into VP4 and VP2 in HAV particles?

Analysis of HAV particles reveals that even in RNA-containing full particles, VP0 is only partially cleaved, with VP4 detection possible as in other picornaviruses. Empty particles contain only uncleaved VP0 and are likely similar to empty particles frequently observed in picornavirus infections . Full HAV particles appear to contain more uncleaved VP0 than typically seen in other picornaviruses, consistent with observations that VP0 cleavage is "protracted" in HAV . This characteristic processing pattern may be related to HAV's unique assembly and maturation pathway.

How do mutations in VP4 affect viral viability and function?

Studies on VP4 mutations in picornaviruses have revealed critical insights into its function during infection. Mutations at threonine-28 of VP4 produce dramatically different outcomes: glycine substitution (4028T.G) renders the virus nonviable, while valine (4028T.V) and serine (4028T.S) substitutions maintain viability . The nonviable 4028T.G mutant is defective in the viral entry pathway. While VP4 insertion into cellular membranes occurs during initial infection stages for all variants, the 4028T.G mutant fails to deliver the viral genome to the cytoplasm . This failure correlates with an inability to form ion channels in lipid bilayers, unlike the viable mutants. These findings suggest that ion channel formation is essential for successful uncoating and genome delivery, with the VP4 sequence directly contributing to channel architecture .

What does HAV's phylogenetic position reveal about picornavirus evolution?

Structure-based phylogenetic analysis places HAV as an evolutionary link between classical picornaviruses and insect picorna-like viruses . The N-terminal domain swap in VP2 makes it more similar to the homologous VP1 and VP3 proteins, supporting the hypothesis that HAV retains structural and functional features characteristic of primordial picornaviruses that resembled present-day insect picorna-like viruses . This evolutionary positioning suggests that the subsequent acquisition of efficient cell entry mechanisms allowed for the diversification seen in modern mammalian picornaviruses. HAV's enigmatic properties, including its ability to move from cell to cell by transcytosis, may reflect its position as this evolutionary link .

How do typing methods for HAV compare with those used for other enteroviruses?

While VP1 sequencing has become the reference test for typing human enteroviruses (HEVs), researchers have developed alternative approaches targeting other capsid proteins. A reverse transcription-PCR assay targeting the central part of the VP2 capsid protein has proven effective for typing HEVs at the serotype level . This method uses two primer pairs to amplify a fragment of 584 bp or a portion of it (368 bp), with seminested primers enhancing sensitivity for difficult-to-amplify strains . When applied to 116 clinical and environmental HEV strains, the VP2 method correctly identified all 61 typeable isolates at the serotype level when compared to seroneutralization results. For 48 of 55 "untypeable" strains (87.3%), the VP2 method yielded the same serotype identification as VP1 sequencing . Notably, for six strains that could not be amplified by the VP1 method, the VP2 approach successfully provided serotype identification, demonstrating its value as a complementary typing strategy.

What experimental approaches are used to study VP4-mediated membrane penetration?

Researchers employ a combination of biophysical assays and molecular dynamics simulation studies to characterize VP4's membrane-penetrating activity . These methodologies allow for the assessment of VP4's ability to integrate into membrane vesicles and form pores. Membrane disruption is evaluated by measuring leakage from vesicles while monitoring vesicle size and shape. Specifically, researchers use vesicles that mimic the lipid composition of late endosomes under acidic pH conditions to demonstrate the specificity of VP4's membrane activity . Through these approaches, researchers have proposed a mechanism whereby initial penetration of VP4's N-terminus into the outer leaflet of membranes eventually results in the formation of small pores in lipid vesicles, facilitating viral genome release.

How are VP4 mutations generated and characterized in experimental studies?

VP4 mutations are typically created through site-specific mutagenesis, introducing specific amino acid substitutions at targeted positions, such as threonine-28 . The characterization of these mutants involves comprehensive functional analyses, including:

• Viability assessment of the resulting virus

• Evaluation of receptor binding capability

• Analysis of VP4 insertion into cellular membranes

• Monitoring cytoplasmic delivery of the viral genome

• Testing the formation of ion channels in lipid bilayers

• Characterizing the electrical properties of formed ion channels

These methodological approaches have revealed that the ability to form ion channels correlates directly with successful genome delivery and viral viability, highlighting VP4's critical role in the infection process .

What are the implications of HAV VP4-VP2 research for antiviral development?

Understanding the unique structural and functional properties of HAV VP4-VP2 provides potential targets for antiviral development. The membrane-penetrating activity of VP4, despite its atypical structure, represents a critical vulnerability in the viral life cycle that could be exploited therapeutically . Additionally, the unusual stability of HAV particles conferred by specific interactions at the icosahedral 2-fold axes suggests possible approaches to destabilize the virus . Further research into the structure-function relationships of these capsid proteins could lead to the development of novel antivirals that specifically disrupt HAV capsid assembly, stability, or membrane penetration activities.

How might further characterization of HAV antigenic sites inform vaccine development?

Structure-based predictive methodology has identified potential antigenic sites on HAV, including VP2 residues 71 and 198, and VP3 residues 89-96 . Further characterization of these epitopes could enhance our understanding of HAV immunogenicity and inform the development of more effective vaccines. The detailed mapping of antigenic sites on the HAV capsid surface, particularly those involving VP4-VP2 components, would provide valuable insights for rational vaccine design strategies aimed at eliciting robust protective immune responses.

What are the optimal storage and handling conditions for recombinant HAV VP4-VP2?

Recombinant HAV VP4-VP2 should be stored at -20°C for long-term stability . The protein is typically shipped on blue ice and is formulated in 10 mM CBB pH 9.6, 0.1% SDS, and 50% glycerol to maintain stability . When working with this reagent, researchers should follow standard protein handling protocols to minimize freeze-thaw cycles and maintain protein integrity for experimental applications in ELISA and Western blot assays.