Recombinant Australian bat lyssavirus Glycoprotein G (G)

What is Australian Bat Lyssavirus (ABLV) Glycoprotein G and what is its significance in viral research?

Australian bat lyssavirus (ABLV) is a rhabdovirus of the genus lyssavirus that is endemic in Australian bat populations. It causes a neurological disease in humans that is clinically indistinguishable from rabies. The virus has two distinct variants: one circulating in frugivorous bats (genus Pteropus) and another in insectivorous microbats (genus Saccolaimus) .

The glycoprotein G (G) is the single fusogenic envelope protein of ABLV responsible for:

• Viral attachment to host cell receptors

• Fusion with the host cell membrane

• Mediating receptor-mediated endocytosis followed by pH-dependent fusion

• Inducing protective immune responses

From a research perspective, the G protein is significant because it is the only viral protein exposed on the virion surface (forming trimers), making it the primary target for neutralizing antibodies. Understanding its structure and function is critical for developing diagnostic tools, vaccines, and therapeutics against ABLV infections .

How is recombinant ABLV Glycoprotein G produced for research purposes?

Recombinant ABLV G protein can be produced through several methodologies:

Recombinant Vesicular Stomatitis Virus (rVSV) Expression System:

The most widely documented approach uses recombinant vesicular stomatitis viruses (rVSVs) expressing ABLV G glycoproteins. This method involves:

• Construction of a plasmid containing the ABLV G gene

• Replacement of the native VSV G gene with the ABLV G sequence

• Rescue of recombinant virus using reverse genetics techniques

• Amplification in permissive cell lines

• Verification of G protein expression through immunofluorescence or Western blotting

These rVSV-ABLV G constructs often incorporate reporter genes such as maxGFP to facilitate visualization and quantification of viral infection in host cells. This system allows researchers to study ABLV G-mediated entry without requiring BSL-4 facilities needed for wild-type ABLV .

What methods are used to study evolution and recombination events in ABLV Glycoprotein G?

Research on ABLV G evolution employs several computational and experimental approaches:

Computational Methods:

For example, in one comprehensive study, researchers analyzed 53 full-length glycoprotein gene sequences isolated from different hosts in 21 countries over a period of 70 years to investigate recombination events and selection pressures in the G gene of Lyssaviruses .

What evidence exists for recombination events in ABLV and other lyssavirus G proteins?

Recombination events in lyssavirus G proteins appear to be rare but have been documented:

Key Findings:

• In a computational analysis of 53 lyssavirus G gene sequences spanning 70 years and 21 countries, only one potential recombinant was detected: AY987478, a dog isolate of CHAND03 (genotype 1) in India .

• The recombination event was confirmed using multiple detection methods:

  • BootScanning analysis identified two potential breakpoints at nucleotide positions 440-540 and 1000-1130

  • Statistical support showed P-values of 0.0004 for the breakpoints

  • High bootstrap values supported clustering with different parent sequences in different regions of the gene

• When this putative recombinant sequence was excluded from analyses, no further recombination events were detected in the dataset .

This rarity of recombination suggests that the G gene in lyssaviruses has been primarily under purifying selection rather than experiencing frequent genetic exchange events. This finding has implications for our understanding of lyssavirus adaptation mechanisms and vaccine development strategies .

What selection pressures act on ABLV Glycoprotein G and how do they affect viral evolution?

Research indicates that ABLV G protein, like other lyssavirus G proteins, experiences predominantly negative (purifying) selection pressure:

Selection Pressure Findings:

These findings contrast with some other RNA viruses where positive selection drives adaptation, suggesting that ABLV may use alternative mechanisms for cross-species transmission .

How does ABLV Glycoprotein G mediate viral entry into host cells?

ABLV G protein mediates a complex entry process that has been characterized using recombinant expression systems:

Entry Mechanism:

• Initial Attachment:

  • G protein trimers on the virion surface bind to specific cell surface receptors

  • The precise identity of these receptors remains under investigation

• Endocytic Pathway:

  • ABLV enters cells primarily through clathrin-mediated endocytosis

  • Entry requires functional dynamin for vesicle formation

  • Inhibition of clathrin-coated pit formation blocks viral entry

  • Caveolar-dependent endocytosis and macropinocytosis are not significantly involved

• Cytoskeletal Requirements:

  • Actin polymerization is essential for successful ABLV G-mediated entry

  • Disruption of the actin cytoskeleton inhibits viral infection

• Endosomal Trafficking:

  • Rab5, a small GTPase involved in early endosome formation and trafficking, is required

  • Expression of dominant-negative Rab5 mutants blocks ABLV G-mediated entry

  • In contrast, Rab7 (late endosomes) and Rab11 (recycling endosomes) are not essential

• pH-Dependent Fusion:

  • Low pH within endosomes triggers conformational changes in the G protein

  • These changes facilitate fusion between the viral and endosomal membranes

  • This fusion event releases the viral nucleocapsid into the cytoplasm

These findings were established using maxGFP-encoding recombinant vesicular stomatitis viruses expressing ABLV G proteins, allowing for detailed characterization of the entry pathway without requiring work with live ABLV .

What methodological approaches are used to study ABLV G-mediated viral entry?

Researchers employ several sophisticated techniques to investigate ABLV G-mediated entry:

Experimental Approaches:

• Recombinant Virus Systems:

  • maxGFP-encoding recombinant VSVs expressing ABLV G glycoproteins

  • These systems allow visualization and quantification of infection in various cell types

• Pharmacological Inhibitors:

  • Dynasore to inhibit dynamin function

  • Chlorpromazine to disrupt clathrin-coated pit formation

  • Nystatin and methyl-β-cyclodextrin to disrupt caveolae

  • EIPA to block macropinocytosis

  • Cytochalasin D and latrunculin A to disrupt actin polymerization

• Dominant-Negative Mutants:

  • Expression of Eps15 dominant-negative mutants to block clathrin-mediated endocytosis

  • Dominant-negative Rab5, Rab7, and Rab11 mutants to investigate endosomal trafficking requirements

• Confocal Microscopy:

  • Used to visualize virus localization and co-localization with cellular markers

  • Tracking of fluorescently labeled virus particles during entry

• Biochemical Assays:

  • Cholera toxin B subunit uptake assays to confirm disruption of caveolar endocytosis

  • Western blotting to confirm expression of dominant-negative proteins

• Statistical Analysis:

  • Typically using one-way ANOVA with Dunnett's multiple comparisons test

  • Establishing significance thresholds (typically p<0.05)

These methodologies allow for detailed dissection of the molecular mechanisms governing ABLV G-mediated entry, providing insights for antiviral development.

How does the host range and cell tropism of ABLV G compare to other lyssaviruses?

ABLV G protein demonstrates interesting patterns of host range and cell tropism:

Host Range and Tropism:

• Natural Host Spectrum:

  • Two distinct variants of ABLV exist: one in frugivorous bats (pteropid bats - ABLVp) and one in insectivorous microbats (yellow-bellied sheath-tailed bat - ABLVs)

  • ABLV has caused fatal infections in humans

  • Recent evidence shows ABLV can infect horses, demonstrating potential for broader cross-species transmission

• Experimental Cell Tropism Studies:

  • Recombinant VSVs expressing ABLV G glycoproteins have been used to examine tropism across cell lines from various mammalian species

  • ABLV G can mediate entry into cells from diverse mammalian origins, suggesting broad tropism

• Comparative Analysis:

  • ABLV G shares highest sequence similarity with rabies virus (RABV) G and other phylogroup 1 lyssavirus G proteins

  • This similarity correlates with shared entry mechanisms and cross-neutralization patterns

  • Despite different natural host reservoirs, the entry pathways utilized by ABLV G and RABV G are remarkably similar

• Structural Determinants:

  • The conservation of key structural elements in the G protein likely explains the shared tropism

  • Domains involved in receptor binding and fusion activity are particularly conserved among phylogroup 1 lyssaviruses

The broad tropism of ABLV G protein supports its potential for cross-species transmission but other factors likely determine the limited host range observed in natural settings .

What epitopes on ABLV G are recognized by neutralizing antibodies and how can this information be used in diagnostics and therapeutics?

Research on ABLV G epitopes has yielded important insights for antibody development:

Neutralizing Epitopes and Applications:

• Key Epitope Identification:

  • Phage display techniques have been used to identify human monoclonal antibodies (hmAbs) that recognize and neutralize ABLV G

  • Two notable hmAbs, A6 and F11, recognize overlapping epitopes on the lyssavirus G protein

  • These antibodies effectively neutralize phylogroup 1 lyssaviruses including both ABLVs/ABLVp variants and RABV

• Neutralization Potency:

  • A6 and F11 completely neutralize ABLVs/ABLVp at concentrations ranging from 0.39 to 6.25 μg/mL

  • They neutralize RABV at even lower concentrations (0.19 to 0.39 μg/mL)

  • Their effectiveness spans across phylogroup 1 lyssaviruses while having minimal effect on phylogroup 2 and non-grouped diverse lyssaviruses

• Therapeutic Applications:

  • The potent neutralizing activity suggests potential use in post-exposure prophylaxis

  • The cross-reactivity across phylogroup 1 lyssaviruses indicates broader protection than current strain-specific approaches

  • These antibodies could complement or potentially replace rabies immunoglobulin in treatment protocols

• Diagnostic Applications:

  • The epitope specificity allows for development of improved diagnostic assays

  • Could enable differentiation between phylogroup 1 and other lyssavirus infections

  • Potential for developing rapid point-of-care diagnostic tests

This research demonstrates the value of epitope mapping in developing cross-reactive antibodies against ABLV G, with significant implications for both therapeutics and diagnostics .

What are the challenges in expressing and purifying recombinant ABLV G for structural studies?

Researchers face several challenges when producing recombinant ABLV G for structural analysis:

Technical Challenges:

• Protein Expression Systems:

  • Membrane proteins like ABLV G are typically difficult to express in soluble form

  • Expression systems must maintain proper protein folding and post-translational modifications

  • Common approaches include:

    • Viral vector systems (VSV, vaccinia)

    • Mammalian cell expression

    • Truncated soluble forms lacking transmembrane domains

• Structural Constraints:

  • ABLV G undergoes pH-dependent conformational changes critical to its function

  • These conformational states complicate structural determination

  • The protein must be stabilized in a particular conformation for successful structural studies

• Glycosylation:

  • As a glycoprotein, ABLV G contains post-translational modifications

  • Glycosylation patterns can affect protein folding, stability, and antigenic properties

  • Expression systems must reproduce relevant glycosylation patterns for meaningful structural analysis

• Purification Challenges:

  • Membrane proteins require detergent solubilization

  • Finding detergents that maintain native structure while allowing purification is complex

  • Aggregation during concentration steps presents additional difficulties

• Trimeric Structure:

  • Native ABLV G exists as trimers on the virion surface

  • Maintaining this oligomeric state during purification is challenging but essential for studying authentic structures

Researchers have addressed some of these challenges by using recombinant VSV systems expressing ABLV G and by developing soluble G protein constructs that retain antigenic properties while being more amenable to purification and structural studies .