Recombinant Rabbit hemorrhagic disease virus Protein VP2 (ORF2)

What is the genomic organization of VP2 within the RHDV genome?

VP2 is encoded by ORF2, which is located at the extreme 3' end of both the genomic and subgenomic RNAs of RHDV. The start codon for ORF2 is positioned at nucleotide 7025 and shares a 17-nucleotide overlap with ORF1, but has a +2 frame shift relative to the capsid protein (VP60) ORF. This genomic arrangement is characteristic of caliciviruses and facilitates coordinated expression of viral proteins during infection . The genomic positioning suggests a tightly regulated expression mechanism that coordinates with major capsid protein production.

What are the molecular characteristics of VP2 protein?

RHDV VP2 consists of 117 amino acids and encodes a polypeptide of approximately 12.7 kDa . While less abundant than the major capsid protein VP60, VP2 is considered an integral structural component of RHDV virions. Unlike VP60, which forms the bulk of the viral capsid, VP2 appears to serve specialized functions related to viral replication regulation rather than primary structural roles. Structural analysis reveals that VP2 may undergo post-translational modifications that affect its functionality in the viral lifecycle.

How does VP2 differ between RHDV subtypes?

Different RHDV subtypes (classic RHDV, RHDVa, and RHDVb/RHDV2) show genetic variations in their VP2 proteins, though these differences are less extensively characterized than those in VP60. Research indicates that RHDVb/RHDV2 has a closer genetic relationship to Rabbit Calicivirus (RCV) than to classic RHDV isolates . These variations may contribute to the different pathogenicity profiles observed among RHDV subtypes, particularly the expanded host range of RHDV2/b which can infect younger rabbits and additional lagomorph species .

What expression systems are most effective for recombinant RHDV VP2 production?

Based on experimental approaches documented in the literature, three primary expression systems have been successfully employed for VP2 production:

• Baculovirus-insect cell system: This system has been effectively used to express both VP60 and VP2, either individually or co-expressed. The system allows for proper folding and post-translational modifications of VP2, making it suitable for structural and functional studies .

• Mammalian expression systems: These have been utilized with luciferase assay systems to study VP2's regulatory effects on VP60 expression. Such systems are particularly valuable when investigating host-pathogen interactions in a context more closely resembling natural infection .

• In vitro coupled transcription/translation systems: These cell-free systems provide a controlled environment for expressing VP2 and studying its direct effects without cellular interference .

When selecting an expression system, researchers should consider the downstream applications and whether native conformation of VP2 is critical for their studies.

What methodological approaches can be used to study VP2's regulatory functions?

To investigate VP2's regulatory role, researchers have employed various experimental approaches:

• Western blot analysis: For quantifying protein expression levels when VP2 is present or absent .

• Dual-luciferase assays: To measure the effect of VP2 on VP60 expression in vivo by comparing reporter gene activity .

• Real-time RT-PCR: To quantify mRNA levels and determine whether regulation occurs at the transcriptional level .

• DNA-launched reverse genetics systems: These allow for the generation of infectious RHDV with or without VP2, enabling assessment of VP2's role in viral replication and infectivity .

Each methodology provides different insights into VP2 functionality and should be selected based on the specific research question being addressed.

How does VP2 affect VP60 expression during viral infection?

Experimental evidence demonstrates that VP2 downregulates the expression of VP60, the major capsid protein, in vivo. This regulatory effect occurs primarily at the transcriptional level, as confirmed by real-time RT-PCR measurements of mRNA levels . The presence of VP2 results in reduced levels of VP60 transcripts, suggesting a feedback mechanism that may help balance structural protein production during viral assembly. This regulatory capacity likely contributes to effective virus infection by ensuring optimal ratios of viral components during assembly.

Is VP2 essential for RHDV infectivity?

What molecular mechanisms underlie VP2's regulatory function?

The molecular basis for VP2's regulatory function appears to involve transcriptional control mechanisms. Analysis suggests that VP2 may interact with viral or cellular transcription factors that modulate the expression of viral genes, particularly VP60 . While the exact mechanism remains to be fully elucidated, real-time RT-PCR data confirms that the regulation occurs at the transcriptional level rather than through post-transcriptional processes. This transcriptional regulation may help orchestrate the viral replication cycle by ensuring appropriate timing and levels of structural protein production.

How does VP2 function compare with VP60 in the context of viral structure and assembly?

While VP60 serves as the major capsid protein and is essential for virion formation, VP2 appears to have a more specialized regulatory role. Both proteins can be expressed from the subgenomic mRNA, though VP60 is also produced via cleavage of the ORF1-encoded polyprotein . Structurally, VP60 consists of three domains: the N-terminal arm (NTA, aa 1–65), the shell (S, aa 66–229), and the protrusion (P, aa 238–579), connected by a short hinge (aa 230–237) . In contrast, VP2's structure is less extensively characterized but appears to function primarily in regulation rather than structural support.

Unlike VP60, which exhibits significant variation in specific regions (V1-V7) between RHDV subtypes that affects tropism and pathogenicity, VP2 variations between subtypes have not been as thoroughly documented . This suggests that VP2's regulatory function may be more conserved across subtypes compared to the more variable antigenic determinants of VP60.

How do N-glycosylation patterns differ between VP2 and VP60?

Research on VP60 has identified multiple N-glycosylation sites that differ between RHDV subtypes:

• RHDVb/RHDV2 has two unique N-glycosylation sites (aa 301, 362) but lacks three other N-glycosylation sites (aa 45, 308, 474) that are present in classic RHDV and RHDVa VP60 .

• These glycosylation differences may affect viral virulence, as deletions of aa 307 and 308 in RHDV VP60 have been shown to impact pathogenicity .

Comparatively, glycosylation patterns of VP2 are less well-characterized but may similarly contribute to functional differences between RHDV subtypes. The divergence in glycosylation patterns represents an important area for future research to understand subtype-specific pathogenicity.

How can recombinant VP2 be utilized in the development of next-generation vaccines against RHDV?

Recombinant VP2 offers several potential applications in vaccine development:

• Adjuvant properties: VP2 may serve as a molecular adjuvant in subunit or VLP-based vaccines, potentially enhancing immune responses to VP60.

• Immune modulation: Given its regulatory role, VP2 might be engineered to optimize immune responses when included in vaccine formulations.

• Cross-protection strategies: By understanding VP2's conservation across RHDV subtypes (GI.1–RHDV, GI.1a–RHDVa and GI.2–RHDV2/b) , researchers could target conserved epitopes for broader protection.

• Attenuated vaccine development: The regulatory role of VP2 in viral replication suggests that modified VP2 constructs could be used to create attenuated virus strains with controlled replication properties.

The development of DNA-launched reverse genetics systems for RHDV provides a platform for generating VP2 variants to test these vaccine strategies .

What techniques are most effective for studying VP2-host protein interactions?

Several methodological approaches can be employed to investigate VP2's interactions with host factors:

• Co-immunoprecipitation assays: To identify host proteins that physically interact with VP2 during different stages of viral replication.

• Yeast two-hybrid screening: For high-throughput identification of potential protein-protein interactions between VP2 and host factors.

• Mass spectrometry-based proteomics: To characterize the composition of VP2-containing complexes isolated from infected cells.

• Confocal microscopy with fluorescently tagged proteins: To visualize subcellular localization and potential co-localization of VP2 with host factors.

• CRISPR-Cas9 screening: To identify host factors whose deletion affects VP2 function or localization.

These techniques can provide insights into how VP2 interfaces with cellular machinery to regulate viral replication and assembly.

How might functional divergence analysis inform our understanding of VP2 evolution?

Functional divergence analysis has revealed several insights about VP60 evolution that may apply to VP2 as well:

• Putative functional divergence-related sites: Analysis identified 50 sites between classic RHDV and RHDVb, 34 sites between RHDVa and RHDVb, and 21 sites between classic RHDV and RHDVa . Similar analysis of VP2 could reveal evolutionary patterns specific to this regulatory protein.

• Phylogenetic relationships: Classic RHDV isolates, RHDVa, and RHDVb form distinct clades, with RHDVa being more closely related to classic RHDV than RHDVb, while RHDVb shows closer genetic relationship to Rabbit Calicivirus (RCV) . These relationships may reflect co-evolution of regulatory functions in VP2.

• Adaptive diversification: Accumulation of amino acid changes in VP60 appears to be a consequence of adaptive diversification during the evolution of RHDV subtypes . Similar adaptive changes in VP2 may reflect evolving regulatory functions in different host environments.

Understanding these evolutionary patterns could inform strategies for predicting emergence of new RHDV variants and guide development of broadly protective countermeasures.

What are the key considerations when designing cDNA constructs for VP2 expression?

When designing cDNA constructs for recombinant VP2 expression, researchers should consider:

• Codon optimization: Adapting the VP2 coding sequence to the codon usage bias of the expression host can significantly improve protein yield.

• Inclusion of appropriate tags: Addition of affinity tags (His, FLAG, etc.) can facilitate purification while minimizing interference with protein function.

• Expression context: For studying regulatory functions, co-expression with VP60 may be necessary to observe physiologically relevant interactions.

• Frame shift considerations: When expressing VP2 from constructs containing both ORF1 and ORF2, maintaining the natural +2 frame shift relative to VP60 is critical for authentic expression .

• Promoter selection: For in vivo studies using DNA-launched systems, the human cytomegalovirus promoter has been successfully employed to drive RHDV genomic expression .

These design considerations can significantly impact the success of recombinant VP2 expression and subsequent functional studies.

How can reverse genetics approaches be applied to study VP2 function?

DNA-launched reverse genetics systems provide powerful tools for studying VP2 function:

• Gene knockout studies: Complete deletion of ORF2 from full-length RHDV cDNA clones allows assessment of VP2's role in virus infectivity and replication .

• Mutagenesis: Site-directed mutagenesis of specific VP2 residues can identify amino acids critical for regulatory function.

• Domain swapping: Replacing VP2 regions with corresponding sequences from other caliciviruses can help identify domains responsible for species-specific functions.

• Reporter gene insertion: Insertion of reporter genes in place of VP2 can allow real-time monitoring of viral replication and spread.

The demonstrated viability of VP2-deleted RHDV provides a unique opportunity to explore the non-essential but regulatory nature of this protein in greater detail than possible with other caliciviruses where VP2 is essential for infectivity .