Recombinant Vaccinia virus Protein L2 (MVA081R, ACAM3000_MVA_081)

What is Vaccinia virus Protein L2 and what is its fundamental role in viral replication?

Vaccinia virus protein L2 is an essential viral component encoded by the L2R open reading frame that plays a critical role in viral morphogenesis, specifically in the formation of crescent-shaped membrane precursors of immature virions. L2 is one of several vaccinia virus proteins necessary for crescent formation and notably the only one synthesized early in infection . The protein is approximately 10.2 kDa in size, though it migrates as a 6-kDa band on SDS-PAGE relative to standard markers . L2 contains potential membrane-spanning domains and is associated with the detergent-soluble membrane fraction of mature virions, consistent with its role in membrane formation processes .

All chordopoxviruses possess L2 homologs, suggesting an evolutionarily conserved and essential function . Indeed, researchers have been unable to isolate infectious L2R deletion mutants, further confirming its critical importance to viral replication . When L2 expression is repressed in conditional lethal mutants, proteolytic processing of major core proteins and the A17 protein (an essential component of immature virion membranes) fails to occur, indicating an early blockage in viral morphogenesis .

What is the subcellular localization pattern of L2 protein during infection?

L2 protein demonstrates a distinct subcellular localization pattern that corresponds to its function. Confocal microscopy analysis using HA-epitope tagged L2 shows that the protein is distributed throughout the cytoplasm in a reticular manner and colocalizes with calnexin, an established endoplasmic reticulum (ER) marker protein . This colocalization is evident throughout the cytosol as well as in the nuclear envelope .

Importantly, L2 and calnexin appear to more intensely stain the periphery of viral factories (detected by DAPI staining) than their interior, particularly up to 6 hours post-infection . This staining pattern is consistent with the enclosure of factories by ER membrane during this period . Unlike viral proteins with late promoters (such as A11, L1, and A17) that localize predominantly in factories, L2's distribution throughout the cytoplasm likely reflects its early expression pattern .

Higher resolution immunogold labeling and transmission electron microscopy confirm that L2 is present in tubular membranes throughout the cytoplasm, including the perinuclear envelope . Notably, L2-labeled tubular membranes are found near viral crescent membranes and close to the free edges of crescents, supporting its role in crescent membrane formation .

How is L2 protein expression regulated during the viral life cycle?

L2 protein demonstrates a distinctive expression pattern during the vaccinia virus life cycle. Analysis of the DNA sequence upstream of the L2R open reading frame reveals the presence of an early promoter, consistent with genome-wide transcriptome analyses . Western blotting experiments confirm that L2 is expressed as an early protein, with detection as early as 2 hours post-infection .

The protein increases substantially in amount by 6 hours post-infection and then only slightly more during the next 18 hours, indicating rapid early synthesis followed by a plateau . Importantly, L2 synthesis continues in the presence of cytosine arabinoside (an inhibitor of DNA replication), which definitively confirms its classification as an early protein . This early expression is significant because L2 is the only protein necessary for crescent formation that is synthesized early in infection, while other proteins involved in this process are expressed late .

Experiments with inducible L2 mutants suggest that while early expression of L2 is advantageous, it is not absolutely essential. When L2 expression is shifted from its natural early timing to late expression (under T7 promoter control), virus replication shows a delay of several hours compared to wild-type virus but can still occur . This indicates some flexibility in the temporal requirements for L2 protein function.

How does L2 protein contribute to viral membrane formation processes?

A similar labeling pattern is observed for the ER luminal protein disulfide isomerase (PDI), and double labeling confirms the localization of L2 and PDI on the same membranes . This suggests that L2 is involved in recruiting or modifying ER membranes for use in viral crescent formation. When L2 expression is repressed in conditional lethal mutants, crescent membrane formation is severely impaired, with large dense aggregates of viroplasm appearing instead of the normal immature and mature virions .

At 8 hours post-infection in the presence of inducer (allowing L2 expression), immature and mature virions are abundantly visible by electron microscopy . In contrast, when L2 expression is repressed, these structures are rare and are replaced by large, dense aggregates of viroplasm . Interestingly, a minority of these aggregates develop short spicule-coated membranes at their periphery, resembling the beginnings of crescent formation . These membrane segments increase in number at later times, and some immature virions eventually form, suggesting that even minute amounts of L2 can support limited and delayed membrane formation .

What experimental strategies have been most effective for studying L2 protein function?

Several complementary experimental approaches have proven valuable for investigating L2 protein function:

• Conditional Lethal Mutants: Since L2 is essential for viral replication (attempts to replace the L2R gene with DNA encoding EGFP were unsuccessful), researchers constructed an inducible mutant with a conditional lethal phenotype . This inducible system used the bacteriophage T7 promoter, the Escherichia coli lac operator, and IPTG as an inducer to regulate L2 expression . This approach allowed researchers to observe the effects of L2 repression on viral morphogenesis.

• Epitope Tagging: A recombinant vaccinia virus with an HA epitope-tagged L2 protein (vL2-HA) was constructed while retaining the natural promoter . This strategy enabled specific detection of L2 using anti-HA monoclonal antibodies, circumventing issues with cross-reactivity encountered with native L2 antibodies . Importantly, the tagged virus replicated with kinetics similar to wild-type virus, confirming that the epitope tag did not disrupt L2 function .

• Confocal Microscopy and Colocalization Studies: Immunofluorescence analysis with antibodies against the HA tag and cellular markers such as calnexin (ER), β-COP (Golgi), and ERGIC-53 (intermediate compartment) allowed determination of L2's subcellular localization .

• Immunogold Electron Microscopy: Higher resolution visualization using immunogold labeling and transmission electron microscopy provided detailed insights into L2's association with membrane structures, particularly its presence in tubular membranes near growing viral crescents .

• Biochemical Fractionation: Analysis of L2's association with virion membranes was performed through detergent extraction with NP-40 alone or with dithiothreitol, which demonstrated that L2 behaves similar to other membrane proteins .

How can researchers effectively visualize L2 protein localization during infection?

Effective visualization of L2 protein during infection requires addressing specific challenges, particularly the cross-reactivity issues encountered with antibodies against native L2. The most successful approach has been to use a recombinant virus expressing HA-epitope tagged L2 (vL2-HA) under its natural promoter . This strategy enables the use of highly specific monoclonal antibodies against the HA tag.

For optimal visualization, a multi-faceted imaging approach is recommended:

What is known about the topology and structural characteristics of L2 protein?

L2 protein demonstrates a specific membrane topology that is critical to its function in viral morphogenesis. Topological studies indicate that the N-terminus of L2 is exposed to the cytoplasm, while the hydrophobic C-terminus is anchored in the endoplasmic reticulum membrane . This orientation is consistent with the protein's role in recruiting or modifying ER membranes for incorporation into viral crescents.

The protein has a predicted molecular mass of 10.2 kDa, though it migrates as a 6-kDa band on SDS-PAGE relative to standard markers . This discrepancy between predicted and observed molecular weight could reflect unusual structural properties or post-translational modifications. L2 contains a single cysteine residue, and approximately 23% of the protein migrates as a 12-kDa species in the absence of reducing agents, suggesting potential disulfide bond formation, though the significance of this observation remains unclear .

Analysis of the L2 sequence reveals two potential membrane-spanning domains, which is consistent with its association with the detergent-soluble membrane fraction of mature virions . This membrane association is demonstrated by extraction properties similar to known membrane proteins such as L1, distinct from core proteins like A3 .

Interestingly, while L2 is primarily associated with ER membranes and viral crescents during morphogenesis, small amounts of L2 and PDI (protein disulfide isomerase, an ER luminal protein) are detected within immature and mature virions, possibly trapped during assembly . Comparative analysis with other viral membrane proteins suggests that the association of L2 with purified virions is minimal compared to proteins such as A9, consistent with its primary role in crescent formation rather than as a structural component of mature virions .

How does L2 protein interact with other viral and cellular components?

L2 protein engages in critical interactions with both viral and cellular components that facilitate its role in viral morphogenesis. The most significant interaction appears to be with the endoplasmic reticulum, as demonstrated by L2's consistent colocalization with calnexin, an ER-resident protein . This association is observed throughout the cytoplasm and at the perinuclear envelope .

Importantly, L2 does not specifically colocalize with markers for the Golgi apparatus (β-COP) or the intermediate compartment (ERGIC-53), although some overlap in staining is observed . This selective association with the ER suggests that L2 specifically recruits or modifies ER membranes for viral crescent formation.

The interaction of L2 with viral components is evidenced by its presence near viral crescents and close to their free edges . Double labeling with antibodies to L2 and the ER luminal protein PDI shows that both proteins are present on the same membranes, suggesting that L2 may be involved in recruiting ER-derived membranes to sites of viral assembly .

A particularly interesting aspect of L2 function is its influence on other viral proteins. The repression of L2 expression, as well as that of A11 and A17 (two other proteins required for viral crescent formation), profoundly decreases the stability of a subset of viral membrane proteins, including those comprising the entry-fusion complex . This suggests that these unstable membrane proteins may need to directly insert into the viral membrane or be rapidly transferred there from the ER to avoid degradation . L2 may therefore play a role in stabilizing or facilitating the proper localization of these proteins.

What are the implications of L2 research for developing novel antiviral strategies?

Research on the L2 protein offers several promising avenues for antiviral development. As an essential protein for vaccinia virus replication that is conserved across all chordopoxviruses, L2 represents an attractive target for broad-spectrum antipoxviral therapeutics . Several specific features make L2 particularly suitable as an antiviral target:

• Early Expression: Unlike other proteins involved in crescent formation, L2 is expressed early in infection . This early expression makes it a potential target for inhibiting viral replication at an initial stage, before significant virus assembly occurs.

• Essential Function: Attempts to create L2 deletion mutants have been unsuccessful, and conditional lethal mutants demonstrate that L2 repression prevents normal virus replication . This essentiality means that effective inhibition of L2 would likely block virus production.

• Unique Topology: The specific membrane topology of L2, with its N-terminus exposed to the cytoplasm and hydrophobic C-terminus anchored in the ER membrane, could allow for the design of drugs that specifically bind to accessible domains and interfere with L2 function .

• Specialized Role: L2's specific role in recruiting ER membranes for viral crescent formation represents a unique process not shared with host cell functions, potentially allowing for selective targeting with minimal host toxicity .

Potential antiviral strategies might include small molecules that bind to L2 and prevent its interaction with ER membranes, compounds that alter L2's conformation or stability, or peptide inhibitors that mimic L2 interaction domains. Additionally, the research on L2 contributes to our fundamental understanding of poxvirus assembly, which may inform broader antiviral approaches targeting viral morphogenesis.

How do modifications to L2 protein affect viral fitness and morphogenesis?

Modifications to L2 protein have significant and varied effects on viral fitness and morphogenesis, providing insights into its function:

• N-terminal HA Epitope Tagging: The addition of an HA epitope tag to the N-terminus of L2, while retaining the natural promoter, does not disrupt virus replication . The recombinant virus vL2-HA forms plaques indistinguishable in size from wild-type virus and exhibits similar replication kinetics . This suggests that the N-terminus of L2 can accommodate modifications without compromising function, potentially due to its cytoplasmic exposure in the protein's topology.

• Conditional Expression: When L2 expression is placed under the control of an inducible promoter (T7 promoter with lac operator), virus replication becomes dependent on the inducer IPTG . Without IPTG, the virus does not form visible plaques, confirming the essential nature of L2 . Interestingly, there is a dose-dependent relationship between IPTG concentration and virus replication, with increased replication occurring at 5 μM IPTG and reaching a plateau between 50 and 100 μM IPTG .

• Temporal Expression Shifts: Changing L2's expression timing from early (natural) to late (T7 promoter-controlled) results in delayed replication by several hours compared to wild-type virus . This indicates that while early expression of L2 is advantageous for optimal replication kinetics, it is not absolutely essential for virus production .

• Repression Effects: When L2 expression is repressed in conditional mutants, profound defects in viral morphogenesis occur . Instead of normal immature and mature virions, large dense aggregates of viroplasm appear . Some of these aggregates develop short spicule-coated membranes at their periphery, resembling the beginnings of crescent formation, which increase in number over time . This suggests that even trace amounts of L2 can support limited membrane formation, though the process is severely impaired and delayed .

What are the primary technical challenges in studying L2 protein function?

Researchers investigating L2 protein face several significant technical challenges:

• Antibody Specificity Issues: Standard antibodies against native L2 present cross-reactivity problems due to a viral band that is expressed after viral DNA replication . This necessitates alternative approaches such as epitope tagging for specific detection .

• Essential Nature of the Protein: The inability to create viable L2 deletion mutants due to its essential role in viral replication complicates direct functional studies . This necessitates the use of conditional lethal systems, which add complexity to experimental design and interpretation .

• Early Expression Timing: As an early expressed protein, L2 is present before viral factories form, resulting in a diffuse cytoplasmic distribution that complicates the study of its specific role in crescent formation occurring later in infection .

• Low Abundance in Mature Virions: Only small amounts of L2 are detected in purified virions compared to other viral membrane proteins . This low abundance makes study of L2 in the context of mature virus particles challenging and may require highly sensitive detection methods .

• Membrane Protein Purification: As a membrane-associated protein with hydrophobic domains, L2 presents the typical challenges associated with purification of membrane proteins, including maintaining proper folding and function outside of the membrane environment .

• Functional Redundancy Possibilities: The observation that even trace amounts of L2 can support limited membrane formation, albeit delayed and impaired, suggests possible functional redundancy or compensatory mechanisms that may complicate interpretation of phenotypes in conditional mutants .

What emerging technologies might advance our understanding of L2 protein?

Several cutting-edge technologies hold promise for deepening our understanding of L2 protein function:

• Cryo-Electron Microscopy and Tomography: These techniques could provide higher resolution structural information about L2's arrangement within membranes and its interaction with other components during crescent formation. Cryo-ET in particular would allow visualization of L2 in its native context without fixation artifacts.

• CRISPR-Cas9 Genome Editing: While complete knockout of L2 is not viable, CRISPR technology could enable more precise modifications, such as domain-specific mutations or regulated degron systems, allowing for more nuanced functional analysis.

• Super-Resolution Microscopy: Techniques such as STORM, PALM, or STED microscopy could provide enhanced spatial resolution of L2 localization during the dynamic process of crescent formation, potentially revealing previously unobservable details of its distribution and movement.

• Proximity Labeling Proteomics: Methods such as BioID or APEX2 could identify proteins in close proximity to L2 during infection, helping to map its interaction network comprehensively. This approach would be particularly valuable for identifying transient or weak interactions that might be missed by conventional co-immunoprecipitation.

• Live-Cell Imaging with Fluorescent Protein Tags: Development of functional fluorescent protein fusions to L2 that maintain virus viability would allow real-time visualization of L2 dynamics during infection, providing insights into the kinetics and spatial regulation of its activity.

• Single-Molecule Tracking: This approach could reveal the movement patterns of individual L2 molecules during viral morphogenesis, potentially illuminating how L2 participates in the recruitment and modification of ER membranes.

• Advanced Lipidomics: Comprehensive analysis of lipid composition in viral membranes and comparison with ER membranes could provide insights into how L2 might be involved in modifying or selecting specific membrane components for incorporation into viral crescents.