Recombinant Vaccinia virus Protein L2 (VACWR089)

What is the L2 protein of Vaccinia virus and what is its role in viral replication?

The L2 protein is encoded by the L2R gene (VACWR089) of Vaccinia virus and plays a critical role in viral morphogenesis, specifically in the formation of crescent-shaped membrane precursors of immature virions. It is expressed early during infection and remains stable throughout the replication cycle . Despite its early expression pattern (unusual for morphogenesis proteins), L2 is essential for virus assembly and is required for the formation or elongation of crescent membranes that eventually form the viral envelope . All chordopoxviruses contain L2 homologs, suggesting its evolutionary conservation and functional importance .

What is the molecular structure and localization of the L2 protein?

The L2 protein has a predicted molecular mass of 10.2 kDa but migrates as a 6-kDa band on SDS-PAGE . It contains two hydrophobic domains in its C-terminal half that function as membrane-spanning regions . 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 (ER) . The protein colocalizes with the ER protein calnexin throughout the cytoplasm of infected cells . L2 is associated with the detergent-soluble membrane fraction of mature virions and can be extracted with NP-40 alone or with dithiothreitol, similar to other viral membrane proteins .

How is L2 protein expression regulated during viral infection?

L2 is expressed early in infection, with detection possible as early as 2 hours post-infection . Its synthesis is not inhibited by cytosine arabinoside (an inhibitor of DNA replication), confirming its classification as an early protein . L2 protein levels increase substantially by 6 hours post-infection and continue to increase slightly over the next 18 hours . This early expression pattern distinguishes it from most other proteins involved in viral morphogenesis, which are typically expressed late in infection . Despite being an early protein, L2 remains stable throughout the infection cycle and becomes incorporated into mature virions .

What approaches can be used to generate recombinant Vaccinia virus with modified L2 protein?

Researchers can generate recombinant Vaccinia viruses with modified L2 proteins through several approaches:

• Epitope tagging: The L2R open reading frame can be replaced with one containing an N-terminal epitope tag while retaining the original promoter . This approach allows for protein tracking without disrupting function.

• Inducible expression systems: Since direct deletion of L2R is lethal, conditional mutants are essential for functional studies. A two-step process can be used:

  • First, insert an inducible copy of L2R (under T7 promoter control with lac operator) into a different locus (e.g., A56R)

  • Then replace the endogenous L2R gene with a marker gene like EGFP

The resulting virus requires an inducer (IPTG) for L2 expression and viral replication . For the construction of plasmids carrying L2R, PCR amplification of the L2R ORF from genomic DNA can be performed using appropriate primers containing restriction sites for insertion into vectors like pVOTE2 .

What methods are effective for detecting and localizing L2 protein in infected cells?

Several complementary techniques can be used to detect and localize L2 protein:

• Western blotting: Using antibodies against L2 (such as those raised against N-terminal peptides) allows detection in cell lysates and purified virions . The protein appears as a 6-kDa band under reducing conditions and may show a 12-kDa species (~23% of total) under non-reducing conditions, suggesting possible disulfide bond formation .

• Immunofluorescence microscopy: For cells infected with epitope-tagged L2, confocal microscopy can visualize L2 colocalization with ER markers such as calnexin throughout the cytoplasm .

• Immunogold electron microscopy: This technique provides higher resolution localization, showing L2 in tubular membranes outside factories, inside factories near crescents, and at the edge or rim of crescents . The labeling pattern resembles that of ER luminal proteins like protein disulfide isomerase (PDI) .

• Subcellular fractionation: L2 can be detected in the detergent-soluble membrane fraction of mature virions, similar to other viral membrane proteins like L1 .

How can conditional mutants of L2 be used to study viral morphogenesis?

Conditional mutants provide powerful tools for studying essential genes like L2R. The repressible L2 system has revealed several key insights:

• Morphogenesis block visualization: When L2 expression is repressed, electron microscopy reveals an accumulation of electron-dense masses of viroplasm without normal crescent formation . Some cells show short membrane-like structures with a "spicule" coat resembling stunted crescents on the exterior of dense masses .

• Protein processing analysis: In the absence of L2 expression, proteolytic processing of the A17 membrane protein is reduced, and processing of A3 and A10 core proteins is prevented, indicating an early block in virus assembly . This can be assessed by pulse-chase experiments with radioactive amino acids followed by SDS-PAGE analysis .

• Quantitative assessment: The degree of repression can be titrated using different IPTG concentrations (5-100 μM), allowing correlation between L2 protein levels and virus replication efficiency .

• Timing studies: The inducible system demonstrates that while early expression of L2 is advantageous (shown by delayed replication kinetics in the inducible system compared to wild-type), expression after DNA replication can still support virus assembly .

What is the relationship between L2 protein and the endoplasmic reticulum during viral morphogenesis?

The association between L2 and the ER provides important insights into the origin of viral membranes:

• Membrane recruitment: L2 appears to participate in the elongation of crescents by facilitating the addition of ER membrane to the growing edge . Immunogold labeling shows L2 present in tubular membranes outside factories and inside factories near crescents, particularly at the edge or rim .

• ER protein incorporation: The similar labeling pattern of L2 and the ER luminal protein PDI suggests that portions of the ER contribute to viral membrane formation . Small amounts of both L2 and PDI are detected within immature and mature virions, possibly trapped during assembly .

• Topological arrangement: L2's N-terminus faces the cytoplasm while its C-terminus is anchored in the ER membrane . This orientation may facilitate interactions with viral and cellular factors during membrane recruitment and crescent formation.

Why might L2 protein be difficult to detect by mass spectrometry despite its essential role?

Despite its essential function, L2 has not been consistently detected in proteomic analyses of purified mature virions . Several factors might explain this apparent contradiction:

• Hydrophobic domains: The presence of two long hydrophobic domains in L2 can complicate extraction and analysis by mass spectrometry . Hydrophobic peptides often have poor solubility in typical mass spectrometry sample preparation buffers.

• Low abundance: L2 may be present in relatively low amounts compared to structural proteins . The small size of L2 (predicted 10.2 kDa) means fewer potential peptides are available for detection after enzymatic digestion.

• Modification or processing: The observed discrepancy between predicted (10.2 kDa) and apparent (6 kDa) molecular weights suggests possible post-translational modification or processing that might affect peptide detection .

• Protein-protein interactions: Strong associations with membrane structures or other viral proteins might reduce extraction efficiency during sample preparation.

How do researchers distinguish between defects in crescent initiation versus elongation when studying L2 mutants?

Interpreting the phenotype of L2-deficient viruses requires careful analysis to distinguish between different possible mechanisms:

• Temporal analysis: Examining infected cells at multiple time points (e.g., 8, 12, 24 hours post-infection) can reveal whether short crescent-like structures appear with delayed kinetics, suggesting an elongation defect rather than initiation failure .

• Quantitative assessment: Counting and measuring the length of membrane segments at the periphery of viroplasm provides quantitative data on the extent of crescent formation .

• Comparison with other mutants: The phenotype of L2-deficient viruses closely resembles that of H7-inducible mutants and shares similarities with A11-inducible mutants, suggesting related functions in membrane biogenesis .

• Leaky expression analysis: Even with conditional systems, minute amounts of protein expression may occur. The appearance of short membrane segments in some L2-repressed cells might be due to minimal L2 expression, allowing initiation but not efficient elongation .

• Complementation studies: Construction of complementing cell lines expressing L2 could potentially distinguish between initiation and elongation defects, though this approach has proven challenging with vaccinia virus proteins .

How does L2 function relate to other viral proteins involved in membrane biogenesis?

Several vaccinia virus proteins participate in membrane biogenesis, but L2 has distinctive features:

This comparison suggests that L2, despite being an early protein, functions cooperatively with late proteins in orchestrating membrane recruitment for virus assembly . The similar phenotypes observed when L2, H7, or A11 are depleted indicate they may function in the same pathway or complex .

What experimental approaches might address remaining questions about L2 function?

Several promising experimental directions could further elucidate L2's precise function:

• Interaction studies: Proteomics approaches like co-immunoprecipitation followed by mass spectrometry could identify viral and cellular proteins that interact with L2, potentially revealing its molecular partners in membrane biogenesis.

• Live-cell imaging: Using fluorescently tagged L2 in combination with markers for the ER and viral factories could enable real-time visualization of membrane recruitment during infection.

• Cryo-electron tomography: This technique could provide higher-resolution structural insights into the precise localization of L2 at the growing edge of viral crescents.

• Domain mutation analysis: Systematic mutation of L2's hydrophobic domains and N-terminal region could map functional regions required for ER association and crescent formation.

• Complementation with L2 homologs: Testing whether L2 proteins from other poxviruses can complement vaccinia L2 deficiency would reveal the degree of functional conservation across the virus family.