Recombinant Potato mop-top virus Movement protein TGB3

What is Potato mop-top virus TGB3 protein and what is its role in viral infection?

TGB3 is one of three proteins encoded by the triple gene block (TGB) module of Potato mop-top virus (PMTV). PMTV is composed of three single-stranded RNA segments, with RNA3 harboring the triple gene block (TGB1, TGB2, and TGB3) . TGB3 plays a critical role in viral cell-to-cell movement and long-distance systemic transport in infected plants . It functions primarily by facilitating the delivery and localization of viral ribonucleoprotein complexes to plasmodesmata, the intercellular channels connecting plant cells . Unlike TGB1 and TGB2 which can bind ssRNA in a sequence non-specific manner, TGB3 does not possess RNA-binding capabilities, suggesting its role is more specialized in transport and localization rather than directly interacting with viral RNA .

How is TGB3 protein localized within plant cells during viral infection?

TGB3 shows a complex and dynamic pattern of subcellular localization during viral infection:

• Early association: TGB3 initially associates with the endoplasmic reticulum (ER) and colocalizes with TGB2 in motile granules that utilize the ER-actin network for intracellular movement .

• Plasmodesmata targeting: A significant portion of TGB3 accumulates at plasmodesmata, even in the absence of TGB2 . Fluorescence microscopy studies have revealed that TGB3 appears as opposing pairs of fluorescent spots across neighboring cell walls, confirming its plasmodesmatal localization .

• Vesicular structures: Later in the infection cycle, TGB3 is incorporated into vesicular structures, but only in the presence of TGB2 . When mRFP-TGB3 is expressed in PMTV-infected epidermal cells, it incorporates into vesicles through recruitment by virus-expressed TGB2 .

• Cell wall association: TGB3 has also been detected in fractions associated with the cell wall, further supporting its role in intercellular transport .

The precise targeting of TGB3 to plasmodesmata appears to occur relatively late in the infection cycle, typically observed 2-3 cell layers away from the infection's leading edge .

What structural features of TGB3 are essential for its proper function?

TGB3 contains several critical structural elements that dictate its functionality:

• Tyr-based sorting motif: TGB3 possesses a putative tyrosine-based sorting motif that is essential for both ER localization and plasmodesmatal targeting. Mutations in this motif completely abolish both the ER association and plasmodesmatal targeting of TGB3 .

• Membrane association domains: As a membrane-associated protein, TGB3 contains hydrophobic domains that facilitate its interaction with cellular endomembranes, particularly the ER network and membranes surrounding the nucleus .

• Protein interaction interfaces: TGB3 contains domains that enable both self-interaction and heterologous interaction with TGB2, which are crucial for its incorporation into vesicular structures and subsequent movement .

The functional importance of these structural features highlights why TGB3 must maintain certain conserved elements while potentially allowing for genetic variation in other regions.

What is known about interactions between TGB3 and other viral or host proteins?

TGB3 engages in several protein-protein interactions that are crucial for viral movement:

• Self-interaction: Yeast two-hybrid experiments have demonstrated that TGB3 can interact with itself, forming homo-oligomeric complexes that may be important for its function .

• TGB2 interaction: A significant heterologous interaction exists between TGB2 and TGB3, which is critical for the incorporation of TGB3 into vesicular structures . This interaction appears to be essential for the recruitment of viral components to plasmodesmata.

• No direct interaction with TGB1: Interestingly, no direct protein-protein interactions have been detected between TGB3 and TGB1, suggesting that TGB2 may serve as an intermediate between these components .

• Host endocytic machinery: TGB3-containing vesicles are labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway, suggesting interaction with the host endocytic machinery .

• Coat protein interaction: When co-expressed with the viral coat protein (CP), TGB3 can induce a hypersensitive response in plants, suggesting functional interaction between these components that influences symptom development .

These interactions collectively facilitate the coordinated movement of viral components within and between cells during infection.

How do mutations in TGB3's Tyr-based sorting motif affect viral movement and pathogenicity?

Mutations in the Tyr-based sorting motif of TGB3 have profound effects on viral movement and pathogenicity:

• Abolishment of ER localization: When the Tyr-based sorting motif is mutated, TGB3 fails to associate with the endoplasmic reticulum, which is the initial site of viral replication and assembly .

• Loss of plasmodesmatal targeting: These mutations also prevent TGB3 from accumulating at plasmodesmata, which completely disrupts the virus's ability to move between cells .

• Impact on symptom development: Since the proper localization of TGB3 is compromised by these mutations, the virus's ability to establish systemic infection and induce symptoms is severely impaired.

• Disruption of interaction with endocytic pathway: The sorting motif appears to be critical for TGB3's interaction with components of the endocytic pathway, which recent research suggests is important for viral intracellular movement .

Methodologically, these effects can be studied using site-directed mutagenesis to introduce specific changes to the Tyr-based motif, followed by expression of fluorescently tagged mutant proteins in plant cells and observation of their localization patterns using confocal microscopy .

What experimental approaches are used to study TGB3 trafficking and function?

Researchers employ various sophisticated techniques to investigate TGB3 trafficking and function:

• Fluorescent protein fusions: Green fluorescent protein (GFP) or monomeric red fluorescent protein (mRFP) are fused to the N-terminus of TGB3 to track its movement and localization in living plant cells using confocal microscopy .

• Viral vector expression systems: Recombinant viral vectors, such as Tobacco mosaic virus (TMV)-based vectors, are used to express TGB3 fusion proteins in plant tissues, allowing for the study of protein behavior in the context of viral infection .

• Biolistic bombardment: Plasmids encoding fluorescently tagged TGB3 are introduced into plant tissues using particle bombardment, enabling transient expression and visualization of the protein .

• Yeast two-hybrid assays: These assays are employed to identify and characterize protein-protein interactions involving TGB3, including self-interactions and interactions with other viral or host proteins .

• Subcellular fractionation: Biochemical fractionation techniques followed by Western blotting are used to determine the association of TGB3 with different cellular compartments .

• Membrane trafficking inhibitors: Compounds that interfere with specific steps in membrane trafficking pathways are used to dissect the route of TGB3 through the cellular endomembrane system .

• Full-length cDNA clones: Generation of infectious full-length cDNA clones of PMTV enables reliable comparative molecular and pathobiological characterization of individual viral isolates and their TGB3 proteins .

These techniques provide complementary information about the dynamic behavior of TGB3 during viral infection and its interactions with cellular components.

How does TGB3 interact with the endocytic pathway during viral movement?

Recent evidence indicates a novel role for the endocytic pathway in PMTV movement, with TGB3 playing a central role in this process:

• Incorporation into endocytic vesicles: TGB3 is incorporated into vesicular structures that are labeled with FM4-64, a marker for plasma membrane internalization and components of the endocytic pathway .

• TGB2-dependent recruitment: This incorporation depends on the presence of TGB2, which colocalizes with Ara7, a Rab5 ortholog that marks the early endosome .

• Association with endocytic recycling machinery: TGB2 interacts with a tobacco protein belonging to the highly conserved RME-8 family of J-domain chaperones, which are essential for endocytic trafficking in multiple organisms .

• Bidirectional movement: TGB3-containing vesicles show bidirectional movement along the ER and transvacuolar strands, suggesting active transport along the cytoskeleton .

• Plasma membrane recycling: The association with endocytic vesicles suggests that TGB3 may utilize plasma membrane recycling pathways to facilitate viral movement between cells.

This interaction with the endocytic pathway represents a novel mechanism for viral intracellular movement, distinct from the previously established models focusing solely on the secretory pathway .

What is the role of TGB3 in symptom development when co-expressed with coat protein?

The co-expression of TGB3 with coat protein (CP) has significant implications for symptom development during PMTV infection:

• Hypersensitive response induction: The combination of CP and TGB3 induces a hypersensitive response in infected plants, characterized by stunted growth, downward curling, and leaf crumpling .

• Symptom severity variation: Plants expressing TGB3 alone show only mild symptoms, but the combination with CP dramatically increases symptom severity compared to other combinations like CP with TGB1 or CP with TGB2 .

• Specific pathological effects: The CP-TGB3 combination appears to trigger specific cellular defense responses that contribute to the observed symptoms, distinct from the direct effects of viral replication and movement.

• Experimental validation: This relationship has been experimentally verified using a potato virus X (PVX)-based expression system to deliver combinations of PMTV genes to model plants like Nicotiana benthamiana .

These findings suggest that while TGB3's primary role is in viral movement, it also contributes significantly to pathogenesis when acting in concert with the coat protein, potentially through interactions with host defense mechanisms .

How does genetic variation in TGB3 across different PMTV isolates affect its function?

The genetic variation of TGB3 across different PMTV isolates shows interesting patterns with functional implications:

• Diversifying selection: The 8K cistron (TGB3) has been found to be under diversifying selection, suggesting that variation in this gene may provide adaptive advantages under different conditions or in different hosts .

• Geographical distribution of variants: A study of PMTV isolates from Peru (the center of potato domestication) revealed significant divergence in TGB3 sequences, with most Peruvian isolates belonging to a different clade (M-type for mild) than those found in Europe, Asia, and North America (S-type for severe) .

• Functional consequences: These genetic variations correlate with differences in pathogenicity, with S-type isolates generally causing more severe symptoms than M-type isolates .

• Recombination events: Evidence for recombination between different TGB3 lineages has been found, suggesting that genetic exchange contributes to TGB3 diversity .

This genetic variation may reflect adaptations to different host plants, vectors, or environmental conditions, and understanding these variations is crucial for developing effective control strategies for PMTV.

What methods are used to produce and purify recombinant TGB3 protein for experimental studies?

Producing and purifying recombinant TGB3 protein involves several specialized techniques:

For experimental applications requiring high purity, recombinant full-length PMTV TGB3, His-tagged protein is commercially available . These purified proteins enable detailed biochemical and structural studies that would be difficult to perform in the context of viral infection.

How can researchers visualize and track TGB3 movement in real-time during infection?

Real-time visualization and tracking of TGB3 movement during infection employs several advanced microscopy techniques:

• Live-cell confocal microscopy: Using GFP or mRFP fusions with TGB3 enables real-time observation of protein localization and movement in living plant cells .

• Photoactivatable fluorescent proteins: These allow for pulse-chase experiments to track the movement of specific populations of TGB3 molecules from their site of synthesis to their final destination.

• FRAP (Fluorescence Recovery After Photobleaching): This technique can measure the mobility of TGB3 within specific cellular compartments by bleaching fluorescence in a defined area and measuring the rate of recovery .

• Split-GFP complementation: This approach can visualize TGB3 interactions with other viral or host proteins in real-time by fusing complementary fragments of GFP to the proteins of interest.

• Multi-channel imaging: Simultaneous imaging of TGB3 along with markers for different cellular compartments (e.g., ER, Golgi, early endosomes) can track its movement through the endomembrane system .

• Time-lapse microscopy: Sequential imaging over time reveals the dynamics of TGB3 trafficking, including rates of movement and pausing at specific cellular locations.

These approaches have revealed that TGB3 moves along the ER and transvacuolar strands, with some populations remaining stationary at the periphery of cells, particularly at plasmodesmata .

What experimental challenges exist in studying TGB3, and how can they be overcome?

Researchers face several specific challenges when studying TGB3:

• Membrane association: TGB3's tight association with membranes can complicate biochemical analyses and structural studies. This can be addressed by using appropriate detergents for solubilization or membrane-mimicking systems for in vitro studies.

• Low expression levels: The natural expression levels of TGB3 during infection are relatively low, making detection challenging. Overexpression systems or highly sensitive detection methods like immunogold labeling for electron microscopy can help overcome this limitation .

• Functional redundancy: Some functions of TGB3 may be partially redundant with other viral proteins, complicating the interpretation of knockout experiments. Complementation assays and careful phenotypic analysis can help disentangle these effects.

• Host range limitations: PMTV has a relatively narrow host range, limiting the model systems available for study. Using heterologous expression systems or developing new experimental hosts can expand research possibilities .

• Distinguishing direct and indirect effects: Determining whether phenotypic effects are directly caused by TGB3 or are downstream consequences of its interaction with other viral components can be difficult. Using inducible expression systems and time-course studies can help establish causality.

• Genetic variability: The genetic variability of TGB3 across different PMTV isolates can complicate comparative studies. Generating full-length cDNA clones of different isolates enables controlled comparison of variants .

By addressing these challenges through methodological innovations and careful experimental design, researchers can continue to unravel the complex functions of TGB3 in PMTV infection.