IBV-NP

What is the structural conformation of IBV-NP in viral ribonucleoprotein complexes?

The helical ribonucleoprotein (RNP)-like structure of IBV-NP reveals a parallel double-stranded conformation, which enables visualization of specific NP-NP and NP-RNA interactions. According to cryo-electron microscopy (cryo-EM) data, each asymmetric unit (NP monomer) relates to adjacent units by a rise of 24.3 Å and a twist of 57.4°, resulting in a right-handed helical reconstruction with approximately 6.3-NP protomers per helical turn. Importantly, research demonstrates that NP alone is insufficient to form the helical structure but requires RNA at the NP-NP interface, which provides crucial insights into the assembly mechanism of the influenza nucleocapsid .

How does the C-terminal region of IBV-NP contribute to its function?

The flexible C-terminus of IBV-NP plays a critical role in oligomerization and RNA binding. Cryo-EM studies show that the last eight C-terminal residues (residues 490-498) are not visible in helical RNP structures where oligomerization occurs, while they are entirely visible in structures where oligomerization is impaired. In the monomeric state, the C-terminal tail makes direct interactions with residues R150, R152, and R355 at positions where RNA density is observed in RNP-like structures. This evidence suggests that IBV-NP undergoes substantial conformational remodeling when transitioning from a monomeric state to a helical RNP state .

What methods are most effective for resolving the high-resolution structure of IBV-NP?

For near-atomic resolution of IBV-NP structures, current research indicates that cryo-EM represents the gold standard approach. The methodology typically involves:

• Initial screening and preliminary imaging using a 200-kV transmission electron microscope (e.g., Talos Glacios)

• Final data acquisition on a higher-powered 300-kV TEM (e.g., Titan Krios)

• Refinement using large datasets of overlapping segments (>200,000 segments)

This approach has enabled researchers to achieve subnanometric structures of IAV NP-RNA complexes in helical conformation at resolutions of approximately 8.7 Å, allowing detailed visualization of protein-protein and protein-RNA interactions .

How does IBV-NP interact with Influenza A virus components during heterotypic co-infection?

In heterotypic co-infection scenarios, IBV-NP (NPB) can inhibit IAV polymerase activity through a specific molecular mechanism. NPB binds directly to its type A counterpart (NPA), disrupting the essential interaction between NPA and PB2. This disruption prevents the formation of functional IAV polymerase complexes, ultimately leading to growth suppression of co-infecting IAV. This mechanism represents one aspect of the phenomenon known as intertypic or heterotypic interference between influenza viruses .

What is the unexpected effect of IAV on IBV replication during co-infection?

Contrary to the conventional understanding of viral interference, recent research using fluorescent reporter-expressing recombinant viruses has revealed that IBV infection is significantly enhanced upon co-infection with IAV. This enhancement is particularly pronounced when IAV infection precedes IBV infection by one hour. The enhancing effect depends specifically on transcription/replication of the IAV genome, as demonstrated through experiments with UV-inactivated IAV and type-specific antiviral compounds. These findings challenge previous assumptions about mutual interference between influenza types and suggest complex interactions that may involve IAV proteins enhancing IBV genome expression or complementing IBV defective particles .

What protocols are recommended for generating recombinant fluorescent reporter IBV for co-infection studies?

For generating recombinant fluorescent reporter IBV strains (such as IBV-mCherry), researchers have successfully employed the following protocol:

• Transfect HEK293T or MDCK cells with bidirectional plasmids containing IBV segments (pDP2002-Bris-PB2, -PB1, -PA, -HA, -NP, -NA, -NS, -M) plus additional plasmid pCI-Bris-PB2

• Use 1 μg of each plasmid with 24 μL of FuGENE® HD and 76 μL of Opti-MEM®

• Incubate cells at 35°C for 24 hours

• Wash cells twice with DMEM and incubate in DMEM containing 1 μg/mL of TPCK-treated trypsin for 48 hours at 35°C

• Harvest and clarify supernatants by centrifugation (5 min at 2,500 g)

• Aliquot and store at -80°C

This methodology enables the creation of fluorescently labeled viruses that maintain replication competence while allowing for precise tracking during infection and co-infection experiments .

How can IBV-NP RNA be quantified accurately in experimental settings?

For precise quantification of IBV-NP RNA in experimental samples, RT-qPCR represents the standard methodology. A recommended protocol includes:

• Extract viral RNA using QIamp Viral RNA kit (Qiagen) from viral stocks

• Perform RT-qPCR using SuperScript III Platinium One-Step qRT-PCR kit

• For IBV detection, target the HA segment using specific primers and probes:

  • Forward primer: 5′-ACCCTACARAMTTGGAACYTCAGG-3′

  • Reverse primer: 5′-ACAGCCCAAGCCCAAGCCATTGTTG-3′

  • Probes: 5′(FAM)-AAATCCAATTTTRCTGGTAG-(BHQ-1)3′ and 5′(FAM)-AATCCGATTTTRCTGGTAG-(BHQ-1)3′

• Generate standard curves using in vitro-transcribed IBV-HA vRNAs

• Run the reaction on a Light Cycler480 instrument (Roche) with the following program: 45°C for 15 min, 95°C for 3 min, 50 cycles of 95°C for 10s, 55°C for 10s, and 72°C for 20s, then 40°C for 30s

This methodology provides sensitive and specific quantification of viral RNA for comparative studies .

What mathematical models best describe IBV-NP behavior in co-infection scenarios?

Mathematical modeling of IBV in co-infection scenarios requires specialized approaches that account for the unique dynamics of viral interference and enhancement. Recent research has developed models covering different aspects of co-infection:

• Intracellular level interference dynamics

• Population level interactions

• Continuous cultivation system behavior

When adapting existing models (such as those for defective interfering particles) to study IBV-NP behavior, researchers must introduce specific modifications including:

• Introduction of specific viral segments (e.g., S7-OP7) as additional segments in the system

• Separate consideration of viral proteins from different strains (e.g., M1-STV and M1-OP7)

• Differentiation between viral ribonucleoprotein complexes bound by different viral proteins

These adjustments are essential because virus infection dynamics differ substantially depending on the cell line used and whether the virus is adapted to that specific cell line .

How is IBV-NP being utilized in nanoparticle-based vaccine approaches?

IBV-NP offers promising applications in next-generation influenza vaccine design, particularly as a structural scaffold for nanoparticle vaccines. Recent research has explored a strategy where:

• A modified B-HA stalk protein is cross-linked onto an IBV nucleoprotein (B-NP) core

• This construct is delivered as a protein nanoparticle

• The nanoparticle vaccine induces class-switched IgG antibodies directed toward the B-HA stalk

• It also generates modest cell-mediated immune responses

In preclinical testing, mice vaccinated with this B-NP-based nanoparticle demonstrated protection from lethal challenge with strains from both B/Victoria and B/Yamagata lineages. This promising approach supports further investigation into stalk-directed IBV vaccines using B-NP as a core structural component .

What role does IBV-NP play in mRNA-based universal influenza vaccine development?

IBV-NP has emerged as a key component in multivalent mRNA vaccine formulations targeting universal protection against influenza viruses. Recent development efforts include:

• Pentavalent mRNA-LNP formulations containing lineage-specific B-HA, B/Colorado/6/2017 (CO/17) IBV neuraminidase (B-NA), B-NP, and matrix protein 2 (B-M2)

• Intradermal delivery systems for these formulations

When delivered either as a single antigen or as part of a multivalent vaccine, these B-NP-containing mRNA formulations have demonstrated ability to induce both neutralizing and non-neutralizing antibody responses in mice. This represents a promising direction for universal influenza vaccine development that might provide broader protection against evolving IBV strains .

What are the comparative advantages of targeting IBV-NP versus other viral proteins in vaccine design?

When designing IBV vaccines, researchers must consider the relative advantages of targeting different viral proteins. The table below compares key features of IBV-NP as a vaccine target versus other viral components:

IBV-NP's high conservation makes it attractive for vaccines targeting cross-protection, though its primarily T-cell mediated responses may require combination with other components for optimal protection .

How does the mechanism of IBV nucleocapsid assembly differ from IAV?

The assembly of the IBV nucleocapsid along one strand involves specific NP-NP and NP-RNA interactions that may differ from the well-characterized IAV assembly process. Based on cryo-EM structures, researchers have proposed that:

• RNA is required at the NP-NP interface for proper helical structure formation of IBV nucleocapsids

• IBV-NP undergoes substantial conformational remodeling when transitioning from monomeric to helical RNP states

• The C-terminal region plays a critical role in this transition, with different conformations observed between oligomeric and monomeric states

These mechanistic insights highlight key differences that may explain why, despite structural similarities between influenza viruses, intertypic reassortments between IAV and IBV have never been detected in nature or successfully generated in vitro. These differences likely stem from incompatible protein functions and incompatible packaging signals between the IAV and IBV vRNPs .

What are the molecular determinants of heterotypic viral interference between IBV-NP and IAV components?

The molecular basis for heterotypic interference between IBV and IAV involves several specific interactions:

• IBV-NP (NPB) directly binds to IAV-NP (NPA)

• This binding disrupts the critical interaction between NPA and PB2

• The disruption prevents formation of functional IAV polymerase complexes

• This leads to suppression of IAV replication in co-infected cells

Additional mechanisms may contribute to this interference, including:

• Inefficient assembly or functionality of heterotypic polymerase complexes

• Competition for cellular resources necessary for replication

• Differential activation of innate immune responses

Understanding these molecular determinants is essential for predicting viral behavior during co-circulating epidemics and may inform antiviral development strategies .

How can structural information about IBV-NP be leveraged for rational antiviral drug design?

The structural insights from cryo-EM studies of IBV-NP provide valuable opportunities for structure-based drug design. Researchers can leverage this information through several approaches:

• Target the RNA-binding groove of IBV-NP with small molecules that compete with viral RNA

• Design compounds that stabilize the monomeric form of IBV-NP, preventing oligomerization and RNP formation

• Develop peptide mimetics that disrupt critical NP-NP interactions required for nucleocapsid assembly

• Create allosteric inhibitors that lock IBV-NP in conformations incompatible with functional RNP formation

Specific structural features that represent promising targets include:

• The interaction interface between C-terminal residues 490-498 and residues R150, R152, and R355

• NP-NP contact points revealed in the helical nucleocapsid structure

• Conformationally flexible regions that undergo remodeling during assembly

Rational drug design using these structural insights could lead to novel antivirals with specific activity against IBV infections, potentially addressing the current limitations in influenza B therapeutics .

What are the implications of IAV enhancement of IBV replication for future pandemic preparedness?

The unexpected enhancement of IBV replication by IAV during co-infection has significant implications for pandemic preparedness strategies. Future research and surveillance should consider:

• Monitoring co-circulation patterns of IAV and IBV strains with enhanced focus on seasons with high prevalence of both types

• Developing improved diagnostic tools capable of detecting co-infections accurately

• Investigating whether this enhancement translates to increased virulence or transmissibility in vivo

• Assessing whether certain IAV strains (particularly pandemic candidates) have greater capacity to enhance IBV replication

• Evaluating whether current antiviral strategies are effective against co-infections versus single infections

These considerations are particularly important given the finding that co-infection frequency increased to 1.3% during the 2017 season with high A(H3N2) and IBV co-circulation, creating conditions that could potentially lead to more complicated clinical outcomes .

How might IBV-NP contribute to next-generation universal influenza vaccine platforms?

IBV-NP holds significant potential for next-generation universal influenza vaccine development through several promising approaches:

• As a protein nanoparticle core for display of conserved epitopes from multiple influenza strains

• As a component in multivalent mRNA-LNP formulations targeting both arms of adaptive immunity

• As a target for inducing cross-reactive T-cell responses that might complement antibody-based protection

Future research directions should focus on:

• Optimizing IBV-NP presentation in various vaccine platforms

• Evaluating the durability of immune responses elicited by IBV-NP-targeted vaccines

• Investigating the potential for IBV-NP to induce trained immunity or other innate immune mechanisms

• Assessing whether targeting IBV-NP can overcome immune imprinting challenges seen with traditional influenza vaccines

Progress in these areas could significantly advance the long-sought goal of developing broadly protective influenza vaccines that reduce the need for annual reformulation .

What computational approaches might best predict IBV-NP evolutionary patterns and inform vaccine design?

Advanced computational approaches for predicting IBV-NP evolution and improving vaccine design should integrate multiple data types and methodologies:

• Sequence-based approaches:

  • Phylogenetic analysis of NP sequences across IBV lineages

  • Identification of selection pressures using dN/dS ratios

  • Epitope mapping and conservation analysis

• Structure-based computational methods:

  • Molecular dynamics simulations to identify functionally important motions

  • Structure-based prediction of antigenic sites

  • In silico mutagenesis to assess evolutionary constraints

• Mathematical modeling approaches:

  • Population-level models of viral evolution

  • Intracellular replication models incorporating NP function

  • Host-pathogen interaction models with immunological components

• Machine learning applications:

  • Prediction of antigenic drift based on NP sequence patterns

  • Identification of key residues that could serve as universal vaccine targets

  • Optimization of multi-epitope vaccine constructs incorporating B-NP components