Recombinant Lagos bat virus Nucleoprotein (N)

What is Lagos bat virus nucleoprotein and what are its primary functions?

The nucleoprotein (N) of Lagos bat virus is one of the five proteins encoded by the lyssavirus genome, alongside the phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L). The N protein serves critical functions in viral replication and pathogenesis by encapsidating the viral RNA genome, protecting it from degradation, and forming part of the ribonucleoprotein complex essential for viral transcription and replication . The high conservation of N protein sequence (100% nucleoprotein identity between isolates from different geographical regions and time periods) suggests it plays a fundamental role in viral fitness and host adaptation . N protein likely contributes to the cooperative effect in pathogenesis observed among lyssavirus proteins, though specific mechanisms are still being elucidated.

What expression systems are most effective for producing recombinant LBV nucleoprotein?

For recombinant expression of LBV nucleoprotein, both prokaryotic and eukaryotic systems have been employed with varying success. E. coli-based expression systems using pET vectors with 6xHis tags provide high yield but may result in inclusion bodies requiring refolding protocols. Baculovirus-insect cell systems offer superior folding and potential post-translational modifications. When designing expression constructs, researchers should consider codon optimization for the host system and inclusion of appropriate purification tags that don't interfere with protein function .

The protocol should include:

• Gene optimization and cloning into an appropriate expression vector

• Transformation/transfection of host cells

• Induction of protein expression (typically with IPTG for bacterial systems)

• Cell lysis under conditions that preserve protein structure

• Purification via affinity chromatography

• Verification of protein identity by Western blot using anti-N antibodies

• Assessment of proper folding through circular dichroism or functional assays

How can researchers confirm the antigenic integrity of recombinant LBV nucleoprotein?

Confirming antigenic integrity of recombinant LBV nucleoprotein requires multiple complementary approaches. First, perform Western blot analysis using specific antibodies against LBV N protein. Second, conduct ELISA with sera from LBV-infected or vaccinated animals. Third, assess the protein's ability to be recognized by LBV-specific monoclonal antibodies in immunofluorescence assays .

Additionally, researchers can employ a virus neutralization test similar to the one used for confirming LBV G protein expression. While the N protein is not directly involved in neutralization, antibodies against properly folded N protein can be detected in sera from infected animals. The antigenic integrity can be verified using LBV antiserum in immunoassays, comparing reactivity to wild-type LBV nucleoprotein . The high conservation of the N protein (100% nucleoprotein identity between isolates) suggests that antibodies raised against different LBV strains should recognize recombinant N protein with similar efficiency .

How can recombinant LBV nucleoprotein be utilized in developing pan-lyssavirus diagnostic assays?

Developing pan-lyssavirus diagnostic assays using recombinant LBV nucleoprotein requires exploiting the conserved epitopes shared across lyssavirus phylogroups. The nucleoprotein's high conservation (100% identity observed among geographically distant LBV isolates) makes it an ideal target for broadly reactive diagnostics .

Methodologically, researchers should:

• Identify immunodominant epitopes through epitope mapping techniques

• Assess cross-reactivity of anti-N antibodies against multiple lyssavirus species

• Develop ELISA or lateral flow assays using recombinant N as capture antigen

• Create multiplex PCR assays targeting conserved regions of N gene

• Validate assay sensitivity and specificity using panels of diverse lyssavirus isolates

Comparative analysis should include testing against phylogroup I lyssaviruses (including classical rabies virus) and other phylogroup II viruses like Mokola virus. While studies have demonstrated that vaccines based on recombinant viruses containing LBV G can induce neutralizing antibodies against both phylogroup I and II lyssaviruses, diagnostic assays based on N protein might offer even broader reactivity due to the protein's highly conserved nature .

What technical challenges arise when studying LBV nucleoprotein interactions with host cellular proteins?

Investigating interactions between LBV nucleoprotein and host cellular proteins presents several technical challenges. The N protein forms complexes with viral RNA and other viral proteins, particularly the phosphoprotein (P), making isolation of N-host protein complexes difficult without disrupting these interactions .

To overcome these challenges, researchers should:

• Use yeast two-hybrid or proximity labeling approaches (BioID, APEX) to identify potential interacting partners

• Confirm interactions through co-immunoprecipitation with appropriate controls

• Visualize interactions in situ using proximity ligation assays or FRET

• Develop cell-free interaction assays using purified components

• Consider RNA-dependency of interactions by including RNase treatments

• Apply cross-linking strategies to capture transient interactions

A significant consideration is distinguishing phylogroup-specific from pan-lyssavirus interactions. Comparative studies with nucleoproteins from RABV (phylogroup I) and LBV (phylogroup II) can reveal unique interaction networks that might explain differences in pathogenicity and host range .

How does LBV nucleoprotein contribute to viral pathogenesis compared to other lyssavirus nucleoproteins?

The contribution of LBV nucleoprotein to viral pathogenesis appears to involve complex mechanisms that may differ from other lyssaviruses. While studies have focused heavily on the roles of G and M proteins in lyssavirus pathogenesis, the N protein's contribution remains less well characterized . Research suggests that N protein may contribute to pathogenesis through:

• Modulation of innate immune responses

• Interaction with host factors affecting viral replication

• Potential role in neurotropism and viral spread

• Influence on viral transcription/replication efficiency

Experimental evidence indicates that viruses containing both LBV M and G genes demonstrate enhanced pathogenicity in mouse models compared to those with only the G gene, suggesting cooperative effects among viral proteins including potential contributions from N protein . When mice were inoculated intracerebrally with various recombinant viruses, those containing LBV components showed differential mortality rates:

These findings suggest that while G protein contributes significantly to pathogenicity, additional viral components including potentially N protein work synergistically to enhance virulence .

What are the optimal methods for studying LBV nucleoprotein's role in RNA encapsidation?

Investigating LBV nucleoprotein's role in RNA encapsidation requires specialized techniques that preserve the nucleoprotein-RNA complex. To effectively study this process, researchers should implement:

• In vitro reconstitution assays using purified recombinant N protein and synthetic RNA

• Electrophoretic mobility shift assays (EMSAs) to assess RNA binding capacity

• Filter binding assays to quantify binding affinity

• Electron microscopy to visualize nucleocapsid-like structures

• Minigenome systems to evaluate functional encapsidation in cells

When designing RNA substrates, researchers should include both specific viral sequences (such as leader regions) and non-specific RNAs as controls. The reconstitution buffer conditions are critical; typically containing 150 mM NaCl, 20 mM Tris pH 7.5, and 1 mM DTT, with variations in salt concentration to assess binding strength .

To quantify encapsidation efficiency, researchers can use RNase protection assays where properly encapsidated RNA remains protected from enzymatic degradation. Additionally, fluorescence-based techniques such as fluorescence anisotropy can provide real-time data on binding kinetics.

How can researchers differentiate between strain-specific and conserved functions of LBV nucleoprotein?

Differentiating between strain-specific and conserved functions of LBV nucleoprotein requires comparative analyses across multiple isolates and related lyssaviruses. The remarkably high conservation observed in LBV nucleoprotein (100% identity between isolates from Senegal, Togo/Egypt, and Kenya) suggests strong evolutionary pressure to maintain critical functions .

A comprehensive approach should include:

• Sequence alignment and phylogenetic analysis of N proteins from diverse LBV isolates

• Structural modeling to identify conserved domains and variable regions

• Site-directed mutagenesis of conserved residues to assess functional impact

• Chimeric protein construction, swapping domains between different lyssavirus N proteins

• Cross-complementation assays using minigenome systems

Experimental data show that LBV isolates maintain extremely high nucleoprotein sequence conservation despite geographical separation and time intervals, with 100% nucleoprotein identity between isolates collected decades apart . This suggests that core functions are highly constrained, while strain-specific functions might be mediated through interactions with other viral proteins that show greater variability.

What are the best approaches for developing serological assays that distinguish LBV nucleoprotein antibodies from other lyssavirus exposures?

Developing serological assays that specifically detect antibodies against LBV nucleoprotein requires careful identification of unique epitopes and validation against sera from different lyssavirus exposures. While nucleoproteins are highly conserved within lyssavirus species, inter-species variations can be exploited for differential diagnosis .

The recommended methodological approach includes:

• Epitope mapping of LBV nucleoprotein to identify unique regions

• Peptide ELISA using strain-specific peptides from variable regions

• Competitive ELISA with LBV-specific monoclonal antibodies

• Protein microarrays featuring recombinant N proteins from multiple lyssaviruses

• Multiplex bead-based assays for simultaneous testing against different lyssaviruses

Validation should involve testing against:

• Sera from confirmed LBV infections/exposures

• Sera from animals infected with other phylogroup II lyssaviruses (MOKV)

• Sera from animals infected with phylogroup I viruses (RABV, DUVV)

• Sera from rabies-vaccinated animals

• Negative control sera

How should researchers address aggregation issues with recombinant LBV nucleoprotein preparations?

Aggregation of recombinant LBV nucleoprotein is a common challenge that can impair functional and structural studies. This tendency toward aggregation likely reflects the protein's natural propensity to self-associate during nucleocapsid formation. To minimize aggregation issues:

• Optimize buffer conditions by screening various pH ranges (typically 7.0-8.5), salt concentrations (150-500 mM NaCl), and additives (glycerol, arginine, low concentrations of non-ionic detergents)

• Express truncated constructs lacking regions prone to aggregation while retaining functional domains

• Co-express with viral phosphoprotein (P) which naturally forms complexes with N and can increase solubility

• Utilize fusion tags that enhance solubility (MBP, SUMO) rather than just purification tags

• Implement size exclusion chromatography as a final purification step to isolate monomeric or defined oligomeric species

• Consider on-column refolding protocols for proteins expressed as inclusion bodies

Dynamic light scattering should be routinely used to monitor aggregation state before proceeding to functional assays. For structural studies, negative-stain electron microscopy can confirm proper assembly of nucleoprotein-RNA complexes versus non-specific aggregates .

What considerations are important when designing primers for LBV nucleoprotein gene amplification from field samples?

Designing effective primers for LBV nucleoprotein gene amplification from field samples requires balancing sensitivity, specificity, and accommodation of potential genetic diversity. Despite the high conservation of LBV nucleoprotein sequence (100% identity observed between some isolates), primer design should account for potential variants .

Key considerations include:

• Target highly conserved regions within the N gene based on multiple sequence alignments

• Design nested PCR protocols to enhance sensitivity for samples with low viral loads

• Include degenerate bases at positions known to vary among LBV strains

• Test primers against related lyssaviruses to ensure specificity or deliberately design pan-lyssavirus primers depending on research goals

• Optimize PCR conditions (particularly annealing temperature and magnesium concentration) for field sample templates that may contain inhibitors

• Include appropriate controls: positive controls from cultured virus, negative field samples, and no-template controls

For field surveillance, researchers should note that nested RT-PCR did not reveal viral RNA in oral swabs of bats in the absence of brain infection, suggesting that surveillance strategies focusing only on oral swabs may underestimate infection rates . When analyzing samples from potential reservoir hosts like Eidolon helvum, consider that seroprevalence can range from 40-67%, providing context for expected detection rates .

How can sequence variations in LBV nucleoprotein genes be correlated with pathogenicity differences?

Correlating sequence variations in LBV nucleoprotein genes with pathogenicity requires integrated bioinformatic and experimental approaches. Despite high sequence conservation (100% identity between some isolates), subtle variations might influence pathogenicity through altered interactions with host factors or other viral proteins .

A comprehensive framework should include:

• Whole-genome sequencing of multiple LBV isolates with documented pathogenicity differences

• Identification of amino acid substitutions in N protein through comparative analysis

• Structural modeling to predict functional impacts of identified substitutions

• Generation of recombinant viruses with specific N protein mutations using reverse genetics

• In vitro growth curve analysis in neuronal cell lines

• Pathogenicity testing in mouse models via both intracerebral and intramuscular routes

• Evaluation of viral spread to various tissues and organs

Research has demonstrated that replacing both M and G genes of an attenuated rabies virus with those from LBV results in increased pathogenicity compared to replacing only the G gene . Similar approaches could be employed to assess N protein contributions by creating chimeric viruses with different combinations of LBV proteins.

Studies with other lyssaviruses have shown that while the G protein is a major determinant of pathogenicity, other viral proteins contribute through cooperative effects . Experimental evidence supports this cooperative pathogenicity model, as recombinant viruses containing both LBV M and G proteins showed 100% mortality in mice after intracerebral inoculation, comparable to wild-type LBV (100%), while those with only LBV G showed lower mortality (75%) .

What are the most promising applications of LBV nucleoprotein in pan-lyssavirus vaccine development?

While current rabies vaccines do not provide cross-protection against phylogroup II lyssaviruses like LBV, research on recombinant viruses expressing LBV proteins suggests potential paths toward pan-lyssavirus vaccines . Though most vaccine development has focused on the G protein due to its role in neutralizing antibody induction, nucleoprotein has potential applications in vaccine design:

• As a component in chimeric virus-like particles (VLPs) displaying epitopes from multiple lyssavirus G proteins

• As a carrier protein for G protein epitopes to enhance immunogenicity

• For inducing T-cell responses that might complement neutralizing antibody responses

• In DNA vaccines expressing both N and G proteins to enhance immunogenicity

Research with SPBNGAS-LBVG-GAS (a recombinant virus with LBV G inserted between two modified RABV G genes) demonstrated induction of virus-neutralizing antibodies against both phylogroup I and II lyssaviruses . The neutralization data showed impressive cross-reactivity:

These results suggest that strategic incorporation of LBV components, potentially including nucleoprotein, could lead to broadly protective vaccines .

How might structural studies of LBV nucleoprotein inform antiviral drug development targeting lyssaviruses?

Structural studies of LBV nucleoprotein could reveal conserved functional domains that represent potential targets for broad-spectrum anti-lyssavirus drugs. The high conservation of N protein sequence across LBV isolates (100% nucleoprotein identity between geographically and temporally distant isolates) suggests critical functional constraints that could be exploited for drug development .

Key approaches should include:

• X-ray crystallography or cryo-electron microscopy of N protein in various states (RNA-free, RNA-bound, P protein-bound)

• Identification of druggable pockets through computational analysis

• Structure-based virtual screening of compound libraries

• Fragment-based drug discovery targeting specific functional domains

• Development of peptide inhibitors that disrupt essential protein-protein interactions

Strategic targets include:

• RNA-binding domain interfaces

• N-P protein interaction sites

• N protein oligomerization interfaces

• Potential allosteric sites that regulate conformational changes

The remarkable conservation of LBV nucleoprotein across Africa suggests that drugs targeting this protein could have pan-African efficacy against LBV variants . Furthermore, by targeting conserved features shared across lyssavirus phylogroups, there may be potential for developing antivirals with activity against both LBV and classical rabies virus.

What can comparative genomics reveal about the evolution and host adaptation of LBV nucleoprotein?

Comparative genomics approaches can provide valuable insights into the evolution and host adaptation of LBV nucleoprotein. The striking conservation of LBV nucleoprotein sequence (100% identity) between isolates from Senegal (1985), France (imported from Togo or Egypt; 1999), and Kenya suggests strong selective pressure maintaining protein function across geographical regions and time .

To further investigate evolutionary patterns:

• Conduct whole-genome sequencing of additional LBV isolates from diverse bat species and geographical locations

• Perform selection analysis to identify sites under purifying versus diversifying selection

• Compare nucleoprotein sequences from bat-associated versus spillover infections in other mammals

• Analyze potential recombination events between different lyssavirus lineages

• Correlate nucleoprotein sequence features with host ranges and ecological niches

The high genome conservancy across space and time suggests that LBV is well-adapted to its natural host species (primarily Eidolon helvum) and that bat populations in eastern and western Africa have sufficient interactions to share pathogens . This has important implications for understanding viral persistence in reservoir populations and potential spillover risks.

The seroprevalence data from different bat colonies (40-67% for E. helvum and 29-46% for R. aegyptiacus) provides context for understanding virus-host dynamics and suggests significant circulation of LBV or related viruses within these populations . Comparative genomics could reveal how nucleoprotein evolution may be shaped by these host population dynamics and transmission patterns.