Recombinant Ovine respiratory syncytial virus Nucleoprotein (N)

What is the genomic organization of ovine RSV and how does the nucleoprotein fit within it?

Ovine respiratory syncytial virus (RSV) belongs to the genus Orthopneumovirus within the family Pneumoviridae and order Mononegavirales. Its genome is a single-stranded, nonsegmented negative-sense RNA of approximately 15,191-15,226 nucleotides . The nucleoprotein (N) encapsidates the entire length of the viral genome to form stable nucleocapsids, which serve as templates for RNA synthesis and remain intact throughout the replicative cycle . N protein is encoded early in the genome sequence, appearing after the NS1, NS2 genes and before the P, M, and SH genes in the standard RSV genome organization .

How does ovine RSV nucleoprotein structure compare with bovine and human RSV counterparts?

Ovine RSV proteins share significant but not identical homology with their bovine and human RSV counterparts. While specific nucleotide sequence identity data for the N gene across all three species isn't explicitly provided in the search results, we can reference comparative data for other proteins. For example, the F gene of ovine RSV has 85% nucleotide identity with bovine RSV and 72-73% with human RSV . Similar patterns of homology likely exist for the N protein. Structurally, the N protein consists of N-terminal and C-terminal domains (NTD and CTD) separated by a flexible hinge region, with the interface between these domains forming the RNA binding groove . Additionally, N possesses N- and C-terminal extensions (N- and C-arms) that are involved in N oligomerization .

What is the role of RSV nucleoprotein in viral replication?

The nucleoprotein plays multiple essential roles in RSV replication:

• Genome protection and structure: N encapsidates the viral RNA genome, protecting it from cellular nucleases and innate immune sensors .

• Replication template: The N-RNA complex serves as the template for the viral polymerase complex (L-P) .

• Viral factory formation: N is involved in forming cytoplasmic inclusion bodies (IBs) or viral factories where viral transcription and replication occur .

• Regulated RNA encapsidation: The switch between monomeric N (N0) and oligomeric N-RNA complexes is crucial for specific encapsidation of viral genomic and antigenomic RNA during replication .

What strategies are used to produce recombinant ovine RSV nucleoprotein for structural studies?

Several approaches can be used to produce recombinant ovine RSV nucleoprotein:

• Bacterial expression systems: Expression of full-length N or truncated N (lacking N- and/or C-terminal arms) in E. coli, often as fusion proteins with tags for purification (e.g., GST) .

• Co-expression with P protein: To obtain monomeric N0 (which otherwise tends to oligomerize and bind RNA non-specifically), co-expression with the N-terminal portion of P protein is effective. For example, researchers have purified a recombinant monomeric N surrogate composed of full-length N and the 40 N-terminal residues of P (N-P40) .

• Mutagenesis approaches: Engineering mutations that prevent RNA binding and/or oligomerization to create Nmono, which mimics the N0 form .

• Genetic code expansion technology: This advanced technique allows site-specific incorporation of non-canonical amino acids into N, enabling precise structural studies through click chemistry-based fluorescent labeling .

The choice of method depends on the specific experimental questions being addressed. Researchers should note that purified N protein is prone to oligomerize upon concentration in the absence of RNA, so stabilization strategies are important .

How can I design a reverse genetics system to generate recombinant ovine RSV with modified nucleoprotein?

A reverse genetics system for generating recombinant ovine RSV requires several components:

• Plasmid construction: Create a full-length cDNA clone of the ovine RSV genome with desired N gene modifications. This typically includes:

  • Plasmids containing support proteins (N, P, L, and M2-1) under control of a T7 promoter

  • A plasmid containing the full-length antigenomic cDNA with desired modifications to the N gene

  • Optional reporter genes (such as GFP) to track infection

• Transfection: Co-transfect cells (typically HEp-2 or BHK cells expressing T7 RNA polymerase) with the plasmids described above .

• Virus recovery: After transfection, incubate cells at appropriate temperature (usually 32-37°C) and harvest virus from the supernatant. Several passages may be required to increase virus titers .

• Verification: Verify the recombinant virus by sequencing and functional assays to confirm that the desired mutations are present and stable .

For ovine RSV specifically, researchers might adapt protocols used for bovine or human RSV, given their genetic similarities .

What assays are available to study N protein-RNA interactions and oligomerization?

Multiple biochemical and biophysical assays can be used to study N protein interactions:

• Surface plasmon resonance (SPR): To measure binding kinetics between N and RNA, or between N and viral proteins like P .

• GST pulldown assays: To identify protein-protein interactions between N and other viral proteins or host factors .

• Size exclusion chromatography (SEC): To analyze the oligomeric state of N protein under different conditions .

• Fluorescence techniques: When using genetically recoded N with non-canonical amino acids, fluorophore labeling allows visualization of N-RNA interactions and assembly dynamics .

• Minigenome assays: To assess the function of N protein in transcription and replication using a reporter gene flanked by viral regulatory sequences .

• Electrophoretic mobility shift assays (EMSA): To analyze N binding to specific RNA sequences.

How does ovine RSV nucleoprotein interact with phosphoprotein (P) and what is the significance of the N0-P complex?

The interaction between ovine RSV nucleoprotein and phosphoprotein is critical for viral replication:

• Chaperone function: The N-terminal region of P binds to newly synthesized N protein, forming an N0-P complex that prevents N from prematurely binding to cellular RNAs and oligomerizing .

• Conformational changes: P binding induces a rotation of NNTD relative to NCTD compared to the oligomeric form. Additionally, the C-terminal arm of N stacks into the positively charged RNA groove, blocking non-specific RNA binding .

• Transition mechanism: The switch from N0-P to N-RNA complexes during viral genome encapsidation is regulated, though the precise mechanisms remain incompletely understood .

• Therapeutic potential: The N0-P interaction represents a potential antiviral target. Studies have shown that overexpression of peptides spanning the N-terminal region of P (e.g., P[1-29]) can inhibit viral RNA synthesis by interfering with the N0-P complex formation .

Research suggests that post-translational modifications of N also play a role in regulating these interactions .

What post-translational modifications regulate ovine RSV nucleoprotein function?

Post-translational modifications (PTMs) play important roles in regulating nucleoprotein function:

• Phosphorylation: Phosphorylation of residue Y88 of N has been validated and shown to modulate N oligomerization. This suggests that RSV N oligomerization depends on regulation by post-translational modifications .

• Other PTMs: While phosphorylation is the most well-characterized modification, other potential PTMs might include ubiquitination, SUMOylation, or acetylation, though these are less well-studied in the context of ovine RSV nucleoprotein .

• Functional consequences: These modifications likely contribute to the spatial and temporal regulation of genome encapsidation, which is critical for efficient RSV replication .

The identification and characterization of these PTMs provide insights into the regulation of viral replication and may reveal new targets for antiviral strategies.

How can we differentiate between ovine, bovine, and human RSV using nucleoprotein-based assays?

Molecular techniques targeting nucleotide or amino acid sequence differences can differentiate between ovine, bovine, and human RSV:

• RT-PCR with restriction enzyme analysis: While the search results describe this approach for the F gene rather than N, a similar strategy could be applied to the N gene. For the F gene, RT-PCR followed by restriction enzyme digestion (EcoRI or MspI) can distinguish between ovine and bovine RSV .

• Sequence-specific primers: Design of primers targeting unique regions of the N gene for each RSV type.

• Serological assays: Using species-specific monoclonal antibodies against N protein epitopes that differ between ovine, bovine, and human RSV.

• Whole genome sequencing: For definitive classification, especially for novel isolates or potential recombinants.

Comparative analysis shows that ovine RSV F gene has 85% nucleotide identity with bovine RSV and 72-73% with human RSV, while the G protein shows only 60% amino acid identity between ovine and bovine RSV . Similar levels of divergence likely exist in the N protein.

What evolutionary insights can be gained from studying ovine RSV nucleoprotein compared to other species?

Studying ovine RSV nucleoprotein in comparison with other species provides several evolutionary insights:

• Classification: The sequence diversity between ovine and bovine RSV nucleoproteins supports their classification as two subgroups of ungulate RSV, similar to how human RSV is classified into subgroups A and B .

• Functional conservation: Despite sequence differences, the core functions of N protein are conserved across RSV species, reflecting evolutionary constraints on proteins essential for viral replication .

• Host adaptation: Differences in N protein sequences may reflect adaptation to different host species and their immune systems.

• Potential recombination: Comparative studies might reveal evidence of recombination events in the evolutionary history of RSV strains.

These evolutionary perspectives can inform our understanding of RSV pathogenesis and host range, with implications for cross-species transmission and vaccine development.

How can recombinant ovine RSV nucleoprotein be utilized in vaccine development strategies?

Recombinant ovine RSV nucleoprotein has several potential applications in vaccine development:

• Live attenuated vaccines: Engineered RSV with modifications to the N gene can generate attenuated viruses that maintain immunogenicity while reducing virulence. For example, recombinant RSV strains with mutations in other genes (like NS1 or M2-2) have shown promise as vaccine candidates .

• Subunit vaccines: Purified recombinant N protein can be used as a subunit vaccine component, potentially in combination with other viral proteins like F or G .

• Nucleic acid vaccines: DNA or mRNA vaccines encoding the N protein could induce cellular immune responses without the risks associated with whole virus vaccines .

• T-cell epitope mapping: Recombinant N protein can be used to identify T-cell epitopes for rational vaccine design, particularly important since N contains conserved epitopes that might provide cross-protection against different RSV strains .

• Safety considerations: Unlike G protein vaccines, which have been associated with enhanced respiratory disease (ERD), N protein-based vaccines might offer a safer alternative, though this requires thorough evaluation .

What approaches can be used to target RSV nucleoprotein for antiviral drug development?

Several strategies can target the RSV nucleoprotein for antiviral development:

• Small molecule inhibitors: Compounds like EDP-938 have been identified as nucleoprotein inhibitors of RSV with potent antiviral activities both in vitro and in non-human primate models .

• Peptide inhibitors: Peptides derived from the N-terminal region of P protein (e.g., P[1-29]) can inhibit viral RNA synthesis by interfering with N0-P complex formation .

• Targeting N oligomerization: Small molecules that disrupt N protein oligomerization could prevent proper nucleocapsid formation and viral replication.

• Interfering with N-RNA interactions: Compounds that prevent N from binding to viral RNA would inhibit genome encapsidation and replication.

• Targeting post-translational modifications: Inhibitors of enzymes responsible for critical N protein modifications, such as kinases that phosphorylate residue Y88, could disrupt viral replication .

These approaches represent promising strategies for developing antivirals with mechanisms distinct from currently available options.

How can we overcome the challenges of studying RSV nucleoprotein structural dynamics during replication?

Studying the structural dynamics of RSV nucleoprotein during replication presents several challenges that can be addressed through innovative approaches:

• Genetically recoded RSV with non-canonical amino acids: This emerging technology allows site-specific incorporation of non-canonical amino acids into N protein, enabling precise fluorescent labeling for visualization of assembly dynamics and trafficking in living cells .

• Cryo-electron microscopy: For capturing different conformational states of N-RNA complexes and nucleocapsid structures.

• Single-molecule techniques: To observe individual N protein molecules during RNA binding and oligomerization in real-time.

• Computational modeling: Molecular dynamics simulations can predict conformational changes in N protein during interactions with RNA and other viral proteins.

• In situ structural studies: Techniques like cryo-electron tomography can visualize viral assembly within cellular contexts.

These methods can provide unprecedented insights into the dynamic processes of nucleocapsid assembly, RNA encapsidation, and viral replication.

What host factors interact with ovine RSV nucleoprotein and how do these interactions influence viral replication?

Understanding host-nucleoprotein interactions opens new avenues for intervention:

• Proteomic approaches: Mass spectrometry-based interactome analyses can identify host proteins that interact with N during different stages of infection.

• Functional genomic screens: CRISPR screens or RNAi approaches can identify host factors essential for N function.

• Immunoprecipitation studies: To validate specific interactions between N and candidate host proteins.

• Cellular localization studies: To determine how host factors influence N localization and inclusion body formation.

• Post-translational modification analysis: To identify host enzymes responsible for modifications of N protein .

These interactions may reveal species-specific determinants of RSV host range and pathogenesis, as well as potential targets for broad-spectrum antivirals.

How does nucleoprotein phosphorylation regulate the formation and function of RSV inclusion bodies?

The role of nucleoprotein phosphorylation in inclusion body dynamics is an emerging area of research:

• Site-specific phosphorylation: Phosphorylation of residue Y88 has been shown to modulate N oligomerization, which likely influences inclusion body formation and organization .

• Kinase identification: Identifying the cellular kinases responsible for N phosphorylation is crucial for understanding regulatory mechanisms.

• Temporal regulation: The timing of phosphorylation events may coordinate the transition between viral transcription and genome replication within inclusion bodies.

• Spatial organization: Phosphorylation may influence the internal architecture of inclusion bodies, potentially creating distinct microenvironments for different viral processes.

• Experimental approaches: Phosphomimetic mutants (e.g., Y88D or Y88E) and phosphodeficient mutants (Y88F) can be used to study the functional consequences of phosphorylation on inclusion body dynamics .

This research area represents a frontier in understanding the spatiotemporal regulation of RSV replication.