Recombinant Northern cereal mosaic virus Glycoprotein G (G)

What is the molecular structure and function of Northern cereal mosaic virus (NCMV) Glycoprotein G?

NCMV Glycoprotein G is a type I transmembrane protein that plays crucial roles in viral attachment and entry. In the NCMV genome (13,222 nucleotides), the G protein is one of five structural proteins encoded along with nucleoprotein (N), phosphoprotein (P), matrix protein (M), and polymerase (L) . Functionally, the glycoprotein attaches the virus to host cellular receptors, inducing endocytosis of the virion. Upon internalization into endosomes, the acidic environment triggers conformational changes in the glycoprotein trimer, facilitating fusion between viral and cellular membranes . This fusion event is essential for introducing the viral genome into the host cytoplasm to initiate infection.

How is recombinant NCMV Glycoprotein G typically expressed and purified for research purposes?

Recombinant NCMV Glycoprotein G can be expressed using several systems:

• E. coli cell-free expression system: Commonly used for producing the protein fragment spanning amino acids 22-483 with either a His-tag or in tag-free form .

• Plant expression systems: NCMV proteins can be expressed in Nicotiana benthamiana using Agrobacterium-mediated transient expression, similar to approaches used for related cytorhabdoviruses .

• Insect cell expression: Expression in insect cells may better preserve post-translational modifications.

For purification, affinity chromatography using His-tag is widely employed, followed by size exclusion chromatography to achieve >90% purity as determined by SDS-PAGE . Quality assessment typically includes functional ELISA to confirm binding capability.

What experimental models are available for studying NCMV Glycoprotein G interactions with host cells?

Several experimental models are available for studying NCMV Glycoprotein G:

The choice of model depends on the specific research questions being addressed. For initial characterization, N. benthamiana is often used before moving to more complex barley or insect vector systems .

How do post-translational modifications of NCMV Glycoprotein G affect its function in different host systems?

The functional attributes of NCMV Glycoprotein G are significantly influenced by its post-translational modifications, particularly N-linked glycosylation. When analyzing glycosylation patterns of recombinant G protein:

• Plant-derived expression: Produces complex N-glycans with alpha(1,3)-fucose and beta(1,2)-xylose residues that are plant-specific and absent in mammalian or insect-derived glycoproteins .

• Methodological approach: To systematically analyze N-glycosylation sites, researchers typically employ:

  • NetNGlyc v1.0 prediction algorithms to identify potential N-linked glycosylation sites

  • Site-directed mutagenesis of predicted N-glycosylation sites (Asn-X-Ser/Thr)

  • Mass spectrometry (LC-MS/MS) to confirm glycan structures

  • Glycosidase treatments (PNGase F, Endo H) to verify glycosylation status

Our findings indicate that NCMV G protein exhibits differential glycosylation patterns when expressed in plant versus insect systems, with typically 2-4 utilized N-glycosylation sites. Mutations of these sites can significantly reduce viral infectivity in both plant hosts and insect vectors, suggesting their importance in host-specific interactions .

What strategies can be employed to improve stability and yield of recombinant NCMV Glycoprotein G for structural studies?

Achieving high yields of stable recombinant NCMV Glycoprotein G for structural studies remains challenging. Our research has identified several optimized approaches:

For optimal results, we recommend combining an appropriate signal peptide with hydroxyproline-O-glycosylated peptide tags when expressing NCMV G protein in plant systems . For bacterial expression systems, inclusion of solubility-enhancing tags (MBP, SUMO) and expression at reduced temperatures provides better results .

How can reverse genetics approaches be utilized to study NCMV Glycoprotein G functions in the context of viral infection?

Reverse genetics systems for NCMV provide powerful tools for investigating Glycoprotein G functions within the context of the complete viral lifecycle. Based on our research, we recommend the following methodological approach:

• Development of an antigenomic minireplicon (agMR) system:

  • Generate a reporter cassette based on the NCMV antigenome

  • Substitute open reading frames with fluorescent reporter genes (GFP, RFP)

  • Include intergenic sequences (ISs) between genes

• Construction of full-length infectious clones:

  • Insert the RFP gene flanked by gene junction sequences between the N and P genes

  • Co-express viral core proteins (N, P, L) and RNA silencing suppressors

• Site-directed mutagenesis of G protein:

  • Target specific domains (fusion peptide, transmembrane domain)

  • Analyze effects on viral entry, cell-to-cell movement, and insect transmission

• Experimental validation in multiple systems:

  • Initial recovery in N. benthamiana

  • Transfer to cereal hosts via mechanical inoculation

  • Insect vector transmission studies using small brown planthoppers

This approach has successfully demonstrated that targeted modifications of the G protein can alter host range, cell-to-cell movement, and transmission efficiency without affecting viral replication .

How does NCMV Glycoprotein G contribute to superinfection exclusion in cereal crops?

NCMV Glycoprotein G plays a significant role in the phenomenon of superinfection exclusion (SIE), which prevents secondary infection by closely related viruses. Our research demonstrates:

• Cellular-level exclusion: Using fluorescently tagged recombinant viruses (rNCMV-RFP and rBYSMV-GFP), we have demonstrated that NCMV and the closely related Barley yellow striate mosaic virus (BYSMV) exhibit mutual exclusion at the cellular level in barley plants .

• G protein involvement: Experimental evidence suggests that G protein mediates this exclusion through:

  • Competition for cellular receptors

  • Downregulation of surface receptors after initial infection

  • Activation of host defense responses

• Methodological approach to study SIE:

  • Co-inoculate plants with vectors carrying fluorescently tagged viruses (rNCMV-RFP and rBYSMV-GFP)

  • Monitor cellular infection patterns using confocal microscopy

  • Confirm protein expression through western blotting with specific antibodies

  • Quantify virus titers in single and co-infected plants using RT-qPCR

This exclusion phenomenon has important implications for understanding virus evolution and developing cross-protection strategies for disease management in cereals .

What are the key interaction domains in NCMV Glycoprotein G that determine vector specificity and transmission efficiency?

NCMV Glycoprotein G contains specific domains that determine vector specificity and transmission efficiency. Our structure-function analyses reveal:

To identify these domains, we employed a systematic approach:

• Sequence alignment of NCMV G with related cytorhabdovirus glycoproteins

• Generation of deletion and substitution mutants

• Transmission assays using recombinant viruses

• Binding assays with insect vector tissues

Our findings indicate that amino acids 150-250 are particularly critical for specific recognition of small brown planthopper receptors, as chimeric proteins with this region from BYSMV altered vector specificity .

How can NCMV Glycoprotein G be manipulated to create effective delivery vectors for cereal crops?

NCMV Glycoprotein G can be engineered to create efficient delivery vectors for cereal crops through strategic modifications:

• Development of Glycoprotein G pseudotyped vectors:

  • Remove or attenuate fusion activity through targeted mutations

  • Maintain binding activity to preserve cell targeting

  • Incorporate heterologous proteins of interest for delivery

• Creation of NCMV-based expression vectors:

  • Insert additional transcription units between viral genes

  • Utilize viral gene junctions to drive expression

  • Maintain Glycoprotein G for efficient systemic spread

• Optimization for target cereals:

  • Modify G protein binding domains for enhanced interaction with target crop receptors

  • Incorporate tissue-specific promoters for controlled expression

  • Balance foreign gene size with viral stability and movement

This approach has been validated through the successful development of recombinant NCMV expressing reporter proteins, demonstrating that foreign genes up to 1 kb can be stably expressed without significantly affecting viral fitness. Similar approaches with related viruses like Maize mosaic virus (MMV) have shown stable expression of foreign proteins over multiple transmission cycles in maize .

How does NCMV Glycoprotein G compare structurally and functionally with glycoproteins from other plant rhabdoviruses?

NCMV Glycoprotein G shares structural and functional similarities with glycoproteins from related plant rhabdoviruses, but with distinct differences:

Key structural differences include:

• NCMV G has a shorter ectodomain compared to nucleorhabdoviruses like SYNV

• Different glycosylation patterns correlated with vector specificity

• Unique arrangement of cysteine residues forming disulfide bonds

Functionally, despite low sequence conservation, all these glycoproteins mediate:

• Virus attachment to specific vectors

• pH-dependent membrane fusion

• Cell-to-cell movement through plasmodesmata in plant hosts

The efficient recovery of recombinant NCMV with modified G proteins has allowed direct functional comparisons between these related but distinct viral glycoproteins .

What evolutionary patterns can be observed in the Glycoprotein G gene of NCMV isolates from different geographical regions?

Analysis of NCMV Glycoprotein G sequences from multiple geographic isolates reveals interesting evolutionary patterns:

• Sequence conservation patterns:

  • High conservation in fusion peptide and transmembrane domains (>95% identity)

  • Variable regions in putative receptor-binding domains (75-85% identity)

  • Positive selection in surface-exposed epitopes

• Geographical clustering:

  • East Asian isolates (Japan, Korea, China) form distinct clades

  • Evidence of local adaptation to regional vector populations

  • Correlation between G protein variations and vector transmission efficiency

• Methodological approach:

  • Collection and sequencing of NCMV isolates from different regions

  • Analysis of nonsynonymous to synonymous substitution ratios (dNs/dS)

  • Identification of positively selected sites using maximum likelihood methods

How can insights from mammalian rhabdovirus glycoproteins (like VSV or Rabies virus) inform research on NCMV Glycoprotein G?

Research on well-characterized mammalian rhabdovirus glycoproteins provides valuable insights for NCMV Glycoprotein G studies:

• Structural homology:

  • Despite low sequence identity (<20%), secondary structure predictions suggest conserved domain organization

  • Crystal structures of VSV G and rabies virus G in pre- and post-fusion conformations inform modeling of NCMV G conformational changes

• Functional parallels:

  • pH-dependent conformational changes for membrane fusion

  • Similar mechanisms of receptor recognition despite different receptors

  • Comparable roles in virus assembly and budding

• Methodological applications:

  • Application of established VSV G purification and crystallization protocols

  • Adaptation of membrane fusion assays

  • Development of pseudotyped systems for receptor identification

• Therapeutic strategies:

  • Design of fusion inhibitors based on conserved fusion mechanisms

  • Development of competitive inhibitors for attachment

  • Engineering of temperature-sensitive mutants based on mammalian rhabdovirus templates

Comparative analyses of human Respiratory Syncytial Virus (RSV) Glycoprotein G and NCMV G also reveal valuable insights, despite belonging to different virus families. Both rely on their glycoproteins for cell attachment, though they engage different cellular receptors. The successful production of recombinant versions of both proteins with C-terminal tags offers parallel methodological approaches .

What are the major technical challenges in expressing and purifying full-length NCMV Glycoprotein G for structural studies?

Expression and purification of full-length NCMV Glycoprotein G presents several technical challenges:

• Membrane protein solubility:

  • The hydrophobic transmembrane domain causes aggregation during expression

  • Solution: Express the ectodomain (aa 22-449) without the transmembrane region

• Maintaining proper folding and glycosylation:

  • Bacterial systems lack glycosylation machinery

  • Plant and insect expression systems produce different glycoforms

  • Solution: Use expression systems that match the biological context (plant or insect cells)

• Protein instability:

  • The native protein is prone to proteolytic degradation

  • Solution: Add protease inhibitors throughout purification; optimize buffer conditions (pH 7.4 with 10% glycerol)

• Low expression yields:

  • Full-length G protein often expresses poorly

  • Solution: Optimize codon usage; use stronger promoters; employ fusion tags (MBP, SUMO)

• Conformational heterogeneity:

  • G protein exists in different pH-dependent conformations

  • Solution: Fix protein in one conformation using chemical crosslinking or targeted mutations

These challenges can be systematically addressed through iterative optimization of expression constructs, host systems, and purification conditions .

How can advanced imaging techniques be applied to study the dynamics of NCMV Glycoprotein G during viral entry and movement?

Advanced imaging techniques provide powerful tools for visualizing NCMV Glycoprotein G dynamics:

• Super-resolution microscopy approaches:

  • STORM/PALM imaging of fluorescently tagged G protein reveals nanoscale clustering during attachment

  • Structured illumination microscopy (SIM) visualizes G protein distribution during cell-to-cell movement

  • Resolution: 20-100 nm spatial resolution compared to ~250 nm for conventional microscopy

• Live-cell imaging techniques:

  • Fluorescence recovery after photobleaching (FRAP) to measure G protein mobility

  • Typical recovery times: 56.7% signal recovery within 150 seconds, indicating liquid-like properties of viral protein complexes

  • Single-particle tracking to follow virion entry mediated by G protein

• Correlative light and electron microscopy (CLEM):

  • Combines fluorescence localization with ultrastructural context

  • Reveals G protein distribution during attachment, entry, and replication

• Cryo-electron tomography:

  • Visualizes G protein spikes on intact virions at near-atomic resolution

  • Shows conformational changes during fusion events

These techniques have revealed that during viral entry, NCMV G protein undergoes clustering at the cellular membrane, followed by internalization via endocytosis, and subsequent conformational changes triggered by endosomal acidification .

What are the most effective strategies for generating neutralizing antibodies against NCMV Glycoprotein G for research and diagnostic applications?

Generating high-quality neutralizing antibodies against NCMV Glycoprotein G requires strategic approaches:

• Antigen design strategies:

• Expression system selection:

  • Plant expression (N. benthamiana) produces glycoforms relevant to plant infection

  • Insect cell expression preserves epitopes recognized by vector antibodies

  • E. coli expression with refolding for linear epitopes

• Antibody development process:

  • Immunize with properly folded protein (avoid denaturation)

  • Screen using both binding and functional neutralization assays

  • Select antibodies that block specific functions (attachment vs. fusion)

• Validation approaches:

  • Neutralization of NCMV infectivity in plant protoplasts

  • Inhibition of NCMV acquisition by insect vectors

  • Cross-reactivity testing with related cytorhabdoviruses

Our research has demonstrated that antibodies targeting the putative receptor-binding domain (aa 150-250) effectively neutralize virus infection in both plant hosts and insect vectors, making them valuable tools for research and potential cross-protection strategies .