Recombinant Vaccinia virus Protein L2 (L2R)

What is Vaccinia virus Protein L2 and what role does it play in viral replication?

Vaccinia virus Protein L2 is an 87-amino-acid, 10.2-kDa protein encoded by the L2R gene. It contains two hydrophobic domains in the C-terminal half and is essential for viral replication . L2 plays a critical role in viral morphogenesis, specifically in the formation of crescent-shaped membrane precursors of immature virions in cytoplasmic factories . It is one of several Vaccinia virus proteins necessary for crescent formation and notably the only one synthesized early in infection . When L2 expression is repressed, viral morphogenesis is blocked at an early stage, demonstrating its essential nature in the viral life cycle .

How conserved is the L2R gene across poxviruses?

Homologs of the L2R open reading frame (ORF) are present in all chordopoxviruses for which sequences have been obtained, indicating an important conserved function . The length of the putative L2-related proteins ranges from 87 to 99 amino acids in representatives of different chordopoxvirus genera . Each homolog maintains a similar structural organization with N-terminal hydrophilic and C-terminal hydrophobic regions . L2 has 96-100% identity to other orthopoxvirus homologs and 31-39% identity to those of other genera of the chordopoxvirus subfamily . This high degree of conservation across all chordopoxviruses suggests an indispensable function in the viral replication cycle.

What is the cellular localization pattern of L2 protein?

L2 protein colocalizes with the endoplasmic reticulum (ER) protein calnexin throughout the cytoplasm of infected and transfected cells . Topological studies have shown that the N-terminus of L2 is exposed to the cytoplasm with the hydrophobic C-terminus anchored in the ER . Using immunogold labeling and electron microscopy, researchers have detected L2 in tubular membranes outside viral factories and inside factories near crescents, particularly close to the edge or rim of crescents . This localization pattern is similar to that of the ER luminal protein disulfide isomerase (PDI) . Small amounts of L2 and PDI have also been detected within immature and mature virions, though these may be trapped during assembly rather than functional components .

How is L2 expressed during viral infection?

L2 is expressed early in infection, confirming its classification as an early viral protein . When researchers infected BS-C-1 cells with the Western Reserve strain of VACV, L2 was detected at 2 hours post-infection as a 6-kDa band (though the predicted mass is 10.2 kDa) . The protein increases greatly in amount by 6 hours post-infection and then only slightly more during the next 18 hours . L2 synthesis occurs in the presence of cytosine arabinoside (an inhibitor of DNA replication), which further confirms its classification as an early protein . After synthesis, L2 remains stable throughout the remainder of the replication cycle and becomes incorporated as a membrane component of mature virions (MVs) .

What experimental approaches demonstrate the essential nature of the L2R gene?

Researchers have employed several sophisticated approaches to establish the essential role of L2R:

• Gene replacement attempts: Initial attempts to replace the L2R gene with DNA encoding Enhanced Green Fluorescent Protein (EGFP) were unsuccessful, providing the first indication that L2 has an essential role in VACV replication .

• Conditional lethal mutant construction: To definitively determine L2's role, researchers constructed a recombinant virus (vL2Ri) in which L2 expression was regulated by the inducer isopropyl β-D-1-thiogalactopyranoside (IPTG) . This approach involved:

  • Starting with vT7lacOI virus (expressing lac repressor regulated by an early/late promoter and T7 RNA polymerase under control of the lac operator and a late promoter)

  • Inserting the L2R gene with a T7 promoter, lac operator, and untranslated leader sequence into the A56R locus

  • Replacing the endogenous L2R gene with EGFP by homologous recombination

• Phenotypic analysis: The resulting vL2Ri virus did not form visible plaques in the absence of IPTG, confirming that L2 expression is essential for replication or virus spread .

• Dose-response studies: Increased replication occurred at 5 μM IPTG and reached a plateau between 50 and 100 μM IPTG, correlating with the induction of L2 protein synthesis shown by Western blotting .

How can researchers create HA-tagged versions of L2 for localization studies?

To overcome limitations with antibody cross-reactivity, researchers can construct recombinant VACV with epitope-tagged L2. A successful approach includes:

• Replace the endogenous L2R ORF with L2R containing an HA epitope sequence at the N-terminus, while retaining the natural promoter .

• Insert a GFP ORF regulated by a late promoter (P11) adjacent to L2R from the same plasmid to facilitate recombinant virus isolation .

• Verify that the tagged protein maintains functionality by confirming normal virus replication kinetics compared to wild-type virus.

This strategy allows for reliable detection of the L2 protein in both biochemical and microscopy studies, overcoming issues with cross-reactive antibodies .

What techniques are most effective for studying L2 protein localization relative to viral structures?

Multiple complementary techniques provide insights into L2 localization:

• Confocal microscopy with fluorescently labeled antibodies against epitope-tagged L2 and cellular markers (such as calnexin for ER) to determine broad cellular distribution .

• Immuno-electron microscopy using gold-labeled antibodies to precisely locate L2 within viral factories and in relation to crescent membranes . This technique revealed that L2 is present near the growing edge of membrane crescents and minimally associated with mature virions .

• Biochemical fractionation to determine membrane association. L2 was found to be associated with the detergent-soluble membrane fraction of mature virions, consistent with its two potential membrane-spanning domains .

• Topological studies to determine protein orientation, which showed that the N-terminus of L2 is exposed to the cytoplasm while the hydrophobic C-terminus is anchored in the ER .

How does L2 repression affect viral morphogenesis?

When L2 expression is repressed in conditional mutants, several critical processes are disrupted:

• Crescent membrane formation: Electron microscopy reveals that immature and mature virions are rare in the absence of inducer and are replaced by large, dense aggregates of viroplasm . Only short spicule-coated membranes resembling the beginnings of crescent formation appear at the periphery of these aggregates .

• Protein processing: Proteolytic processing of major core proteins (P4a/A10R and P4b/A3R) and the A17 protein (an essential component of the immature virion membrane) fails to occur, suggesting an early block in viral morphogenesis .

• Membrane protein stability: Repression of L2, along with 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 these unstable membrane proteins may need to directly insert into the viral membrane or be rapidly transported there from the ER to avoid degradation .

How can researchers distinguish between direct and indirect effects of L2 protein depletion?

Distinguishing direct from indirect effects requires careful experimental design:

• Time-course analysis: Determine the earliest detectable changes after L2 repression. Primary effects should precede secondary consequences.

• Complementation experiments: Supply L2 protein in trans at different timepoints to determine when L2 function is required and if effects can be reversed.

• Control studies with other mutants: Compare phenotypes of L2 conditional mutants with those affecting other proteins involved in crescent formation (like A11 and A17) to identify shared versus unique effects .

• Protein-protein interaction studies: Identify direct binding partners of L2 using techniques such as co-immunoprecipitation, proximity labeling (BioID), or yeast two-hybrid screening.

• Electron microscopy with immunogold labeling: Precisely localize L2 in relation to developing viral structures to correlate its position with observed morphological defects .

What is the significance of L2 being expressed early while functioning in a process that occurs later?

L2's unique status as the only early-expressed protein required for crescent formation raises important research questions:

• The expression timing reflects a crucial preparatory role in morphogenesis. Despite being synthesized early, experimental evidence suggests that de novo synthesis is not required at the earliest stages of infection, as the IPTG-inducible system (which expresses L2 only after DNA replication) still supports virus replication, albeit with a delay .

• This temporal disconnect may indicate that L2 functions in preparing or modifying cellular components (likely ER membranes) well before they are utilized for crescent formation.

• Researchers have noted a delay in the formation of infectious virus when using the inducible system, suggesting that the natural early timing of L2 expression provides an advantage but is not absolutely essential .

• Experimental design to investigate this phenomenon should include precise temporal control of L2 expression with synchronized infections and time-course analyses of membrane modifications and interactions with other viral proteins.

How do the membrane topology and ER association of L2 inform its function?

The specific membrane topology of L2 provides clues to its function:

• The L2 protein has two hydrophobic domains in its C-terminal half, with its N-terminus exposed to the cytoplasm and its C-terminus anchored in the ER membrane .

• This topology positions L2 to potentially recruit or modify ER membranes for incorporation into viral crescents. The cytoplasmic N-terminus could interact with viral or cellular factors while the membrane-embedded portion could facilitate membrane curvature or recruitment.

• L2's colocalization with ER markers throughout the cytoplasm and its detection near the growing edge of crescent membranes suggests it may participate in elongation of crescents by adding ER membrane to the growing edge .

• Researchers can investigate this by designing L2 mutants with altered membrane topology or by disrupting specific domains to determine which regions are essential for function.

How should researchers interpret the apparent size discrepancy of L2 protein?

The L2 protein displays an interesting discrepancy between its predicted and observed molecular weights:

• The L2R ORF is predicted to encode an 87-amino-acid, 10.2-kDa protein, but it migrates as a 6-kDa band on SDS-PAGE .

• Additionally, approximately 23% of the protein migrates as a 12-kDa species in the absence of reducing agent, suggesting possible disulfide bond formation (though this could occur after cell lysis) .

• Researchers should consider several explanations:

  • Post-translational modifications affecting migration

  • Unusual amino acid composition affecting SDS binding

  • Potential proteolytic processing

  • Anomalous migration due to hydrophobic domains

• Verification approaches should include:

  • Mass spectrometry to determine actual molecular weight

  • N-terminal sequencing to check for processing

  • Mutation of the single cysteine to evaluate the role of disulfide formation

  • Expression of epitope-tagged versions at both N- and C-termini

What methodological considerations are important when using inducible systems to study essential viral proteins?

The study of L2 demonstrates several important considerations when using inducible systems:

• Promoter selection: The researchers used a sophisticated system where the lac repressor gene was regulated by the P7.5 early/late promoter, the T7 RNA polymerase gene by the stringent P11 late promoter (and lac operator), and the L2 gene by a T7 promoter (and lac operator) .

• Potential timing issues: This system only works if de novo synthesis of the protein is not required at early times, which was fortunately the case for L2 .

• Leakiness evaluation: Any inducible system should be carefully evaluated for background expression in the uninduced state, as even minute amounts of an essential protein could support some function. In the case of L2, short membrane segments observed at the edge of dense viroplasm increased in number at later times, possibly due to minimal leaky expression .

• Dose-response characterization: Researchers should determine the relationship between inducer concentration and both protein expression and phenotype restoration. For L2, increased replication occurred at 5 μM IPTG and reached a plateau between 50 and 100 μM IPTG .

• Controls: The parental virus (in this case vT7lacOI) should be tested alongside the conditional mutant to confirm that the inducer itself doesn't affect viral replication .

What are promising approaches to further characterize the relationship between L2 and membrane biogenesis?

Several advanced approaches could provide deeper insights:

• Cryo-electron tomography to visualize the three-dimensional structure of membrane crescents with immunogold-labeled L2, allowing precise localization during biogenesis.

• Proximity labeling approaches (BioID or APEX) with L2 as the bait protein to identify proteins in close proximity during viral replication.

• Live-cell imaging with fluorescently tagged L2 to track its dynamics during infection and membrane recruitment.

• Lipidomic analysis comparing wild-type and L2-deficient infections to determine if L2 affects membrane composition.

• Structural studies of purified L2 protein or domains to understand how it interacts with membranes.

• Domain mapping experiments to identify which regions of L2 are required for different aspects of its function (ER localization, crescent formation, protein stabilization).

How might the study of L2 inform broader questions about poxvirus-host interactions?

L2's essential role in viral morphogenesis connects to several broader research questions:

• Membrane remodeling mechanisms: Understanding how poxviruses recruit and reshape host membranes could reveal fundamental principles of membrane biology applicable beyond viral systems.

• ER stress and viral infection: Investigating whether L2's interaction with the ER triggers or modulates ER stress responses could provide insights into how poxviruses manipulate host cell biology.

• Evolution of viral morphogenesis: The conservation of L2 across all chordopoxviruses suggests it represents an ancient and fundamental aspect of poxvirus biology. Comparative studies across diverse poxvirus species could reveal evolutionary constraints and adaptations.

• Antiviral targets: Essential viral proteins with unique functions like L2 represent potential targets for antiviral development. Structure-function studies could inform rational drug design approaches.