Recombinant Variola virus Protein O1 (O1L)

What is Variola virus protein O1 (O1L) and what is its molecular weight?

Variola virus protein O1 (O1L) is a highly conserved orthopoxviral protein encoded by the O1L gene. It is predicted to encode an approximately 78 kDa protein that plays a crucial role in poxvirus pathogenesis . The protein functions as a modulator of the Raf/MEK/ERK signaling pathway in infected cells, complementing the autocrine function of vaccinia virus growth factor (VGF) . Understanding this protein is particularly significant because O1L orthologs are present in a variety of pathogenic orthopoxviruses but notably absent in certain attenuated strains like MVA (Modified Vaccinia virus Ankara) .

What cellular signaling pathways does O1L affect during viral infection?

O1L is required for sustained activation of the Raf/MEK/extracellular signal-regulated kinase (ERK1/2) pathway in infected cells . While the vaccinia virus growth factor (VGF) initially activates this pathway through an epidermal growth factor (EGF)-like mechanism, O1L is necessary to maintain this activation throughout the course of infection . Experimental evidence shows that deletion of the O1L gene in Chorioallantois Vaccinia virus Ankara (CVA) results in only transient ERK1/2 activation after infection, demonstrating its critical role in sustaining this important signaling cascade . The protein appears to function downstream of the epidermal growth factor receptor (EGFR), suggesting a complex interaction with host cell signaling machinery .

How does O1L contribute to viral virulence and pathogenicity?

O1L significantly enhances virulence and pathogenicity through multiple mechanisms:

• Enhanced viral spread: Deletion mutant studies demonstrate that O1L promotes viral spread in infected hosts .

• Increased cytopathic effect: O1L contributes to the cytopathic effect (CPE) in infected cell cultures .

• Sustained ERK1/2 activation: By maintaining ERK1/2 pathway activation, O1L creates a cellular environment more conducive to viral replication .

• Systemic dissemination: In mouse models, O1L enhances spread from lungs to ovaries in intranasally infected BALB/c mice .

Mouse infection experiments clearly demonstrate the virulence role of O1L - when mice were infected with wildtype Western Reserve (WR) vaccinia virus versus O1L deletion mutants, those infected with deletion mutants became sick but recovered to a healthy status, while WR-infected mice experienced significant weight loss resulting in death .

What evidence suggests O1L played a role in the evolution of variola virus host specificity?

Single nucleotide polymorphism (SNP) analysis of orthopoxvirus genomes reveals compelling evidence for O1L's role in host range determination:

• A statistically significant hotspot of genome variation exists within the O1L gene ortholog in variola virus .

• This SNP-dense region is located within a 0.5 kbp segment that maps to the 3′ half of the O1L ortholog .

• Both synonymous and non-synonymous mutations in this region suggest subtle changes to protein function rather than wholesale alterations .

• The pattern of SNPs present in variola virus and its closest relatives (camelpox virus and taterapox virus) points to recombination events that may have contributed to variola's evolution .

The subsequent loss of the functional O1L gene from camelpox virus and taterapox virus, the two closest relatives of variola virus, further suggests that changes within this region of the genome may have played a key role in the evolution of host specificity . Additionally, the absence of an O1L ortholog in MVA isolates, which have restricted host range, provides a potential link between this gene and host range control .

How do O1L sequence variations compare across different orthopoxviruses?

Analysis of O1L sequences across orthopoxviruses reveals significant variation patterns:

The pattern of tolerated SNPs from different groups of cowpox viruses (CPXVs), together with the large number of synonymous SNPs, strongly suggests that recombination events played a significant role in variola virus evolution prior to its split from camelpox and taterapox viruses .

What techniques are used to create O1L deletion mutants for functional studies?

Researchers have employed several approaches to generate O1L deletion mutants:

• Recombinant PCR method: This involves a series of PCR reactions where the enhanced green fluorescent protein (eGFP) gene is amplified and attached to flanking sequences identical to regions on either side of the O1L gene. This construct is then transfected into cells where homologous recombination occurs, replacing O1L with eGFP. The recombinant mutants are subsequently isolated and purified .

• Counterselection cassette approach: As described in one study, the O1L ORF was replaced with an rpsL/neo counterselection cassette. This resulted in the traceless deletion of the O1L ORF and part of the predicted promoter. Removal of the selection cassette was confirmed by sequencing regions flanking the insertion site .

• BAC-based mutagenesis: For reinsertion of a functional O1L gene into MVA (which normally contains a fragmented O1L), researchers used bacterial artificial chromosome (BAC) technology. The fragmented MVA O1L was replaced with an rpsL/neo counterselection cassette, which was then exchanged for the intact CVA version of O1L by allelic exchange .

These methodological approaches allow researchers to study the specific effects of O1L deletion or restoration on viral replication, host range, and pathogenicity.

What are the recommended approaches for detecting O1L protein expression and localization?

Based on the research literature, several approaches have proven effective for studying O1L protein expression and localization:

• Western blot analysis: This technique has been used to determine the size and cellular localization of O1L. Preliminary data indicates that O1L can be detected in the nuclear fraction of cells .

• Immunofluorescence microscopy: This provides an alternative method to investigate the subcellular localization of O1L. The protocol typically involves:

  • Growing cells on coverslips

  • Infecting cell monolayers with wildtype or O1L deletion mutant viruses

  • Fixing and permeabilizing cells with methanol/acetone

  • Blocking with PBS-FBS solution

  • Incubating with anti-O1L sera at various dilutions (1:5000, 1:10000, and 1:50000)

  • Detecting with labeled secondary antibodies

• Generation of specific antibodies: Custom antibodies can be developed by synthesizing peptides from O1L and immunizing rabbits. Multiple injections with adjuvant help develop and maintain the adaptive immune response, resulting in sera containing antibodies specific to O1L .

• Cellular fractionation: This technique helps determine the subcellular compartment where O1L is predominantly located, which provides insights into its function .

How does O1L interact with the NF-κB signaling pathway in infected cells?

While the search results don't directly address O1L's interaction with NF-κB, they do mention another variola virus protein, G1R, that interacts with human nuclear factor kappa-B1 (NF-κB1)/p105 . This represents the first direct interaction between a pathogen-encoded protein and NF-κB1/p105 . Given that O1L affects cellular signaling pathways, future research might explore potential cross-talk between the ERK1/2 pathway modulated by O1L and the NF-κB pathway affected by G1R.

For researchers investigating this question, recommended approaches would include:

• Co-immunoprecipitation assays to detect potential physical interactions between O1L and components of the NF-κB pathway

• Reporter gene assays to measure NF-κB activity in the presence and absence of O1L

• Phosphorylation state analysis of key NF-κB pathway components in cells expressing O1L

• RNA-seq or proteomics analysis to identify changes in NF-κB-dependent gene expression when O1L is present versus absent

What is the mechanism by which O1L sustains ERK1/2 activation during infection?

• O1L complements the function of VGF in sustaining ERK1/2 activation

• O1L seems to function downstream of the EGFR

• The effect is sustained throughout the course of infection

To elucidate this mechanism, researchers should consider:

• Protein interaction studies: Identify binding partners of O1L using techniques such as yeast two-hybrid screening (as was done for variola G1R protein) , co-immunoprecipitation, or proximity labeling approaches.

• Phosphoproteomic analysis: Compare the phosphorylation status of components in the Raf/MEK/ERK pathway between cells infected with wildtype virus versus O1L deletion mutants.

• Domain mapping: Identify functional domains of O1L through mutagenesis studies to determine which regions are critical for ERK1/2 activation.

• Temporal dynamics studies: Investigate the timing of O1L expression in relation to ERK1/2 activation using time-course experiments and synchronized infections.

How does the function of O1L differ between variola virus and other orthopoxviruses?

• Sequence variation: The O1L gene shows significant sequence variation in variola virus compared to other orthopoxviruses, particularly in the 3′ half of the gene .

• Presence/absence patterns: The O1L gene is:

  • Present and functional in variola virus

  • Fragmented in MVA

  • Lost in camelpox virus and taterapox virus, the closest relatives of variola virus

• Host range implications: The pattern of mutations and subsequent loss of O1L in camelpox and taterapox suggests that changes in this gene may have contributed to host range determination .

When investigating these differences, researchers should consider employing chimeric O1L constructs, where domains from different viral O1L proteins are exchanged, to determine which regions are responsible for any functional differences observed.

Can recombinant O1L protein restore virulence to attenuated poxvirus vectors?

This is a critical question for both poxvirus biology and vaccine development. Based on the available information:

These findings suggest that while O1L contributes to virulence, it alone is insufficient to restore full virulence to attenuated vectors like MVA that have multiple attenuating mutations. For researchers exploring this question, it would be important to:

• Test O1L restoration in combination with other virulence factors

• Evaluate O1L's effect on different attenuated poxvirus platforms

• Assess the impact of O1L restoration on immune responses to recombinant antigens expressed from these vectors

• Carefully monitor safety profiles in animal models

What are the implications of O1L research for the development of safer smallpox vaccines?

Understanding O1L function has significant implications for smallpox vaccine development:

• Attenuation strategies: Knowledge of O1L's role in virulence suggests that targeted modification of this gene could contribute to rational attenuation strategies for new vaccine candidates.

• Safety profile improvement: Current approved live smallpox vaccines have significant safety risks . Understanding virulence factors like O1L could help design vaccines with improved safety profiles while maintaining immunogenicity.

• Vector development: For poxvirus-based vaccine vectors expressing heterologous antigens, modulating O1L activity could help optimize the balance between attenuation and immunogenicity.

• Immune response modulation: Since O1L affects cellular signaling pathways, its presence or absence in vaccine strains may influence the quality and magnitude of immune responses generated.

Researchers working on vaccine development should consider evaluating how O1L affects the balance between safety and immunogenicity in preclinical models.

How can knowledge of O1L function inform the development of antiviral strategies against poxviruses?

O1L's role in viral virulence and its effect on cellular signaling pathways suggest several potential antiviral strategies:

• Small molecule inhibitors: Developing compounds that specifically inhibit O1L function could potentially reduce poxvirus virulence without directly affecting virus replication, providing a novel class of antivirals.

• Pathway-targeted approaches: Since O1L sustains ERK1/2 pathway activation, inhibitors of this pathway might show synergistic effects with existing antivirals.

• Combination therapies: Understanding how O1L contributes to pathogenesis could inform the development of combination therapies targeting multiple aspects of the viral life cycle.

• Host-directed therapies: O1L's interaction with host signaling suggests that modulating these pathways could represent a host-directed therapeutic approach less susceptible to viral resistance.

For researchers pursuing these strategies, initial screening could involve evaluating existing ERK pathway inhibitors for their effect on poxvirus replication and pathogenesis in cellular and animal models.