Recombinant Varicella-zoster virus Alpha trans-inducing factor 74 kDa protein (ORF12)

What is VZV ORF12 and what is its role in viral infection?

VZV ORF12 is a tegument protein encoded by the Varicella-zoster virus that plays multiple roles during viral infection. The ORF12 protein is packaged into virions and delivered to cells upon infection, where it immediately begins modulating host cell signaling pathways . Its primary functions include:

• Activation of cellular signaling pathways including PI3K/Akt and MAPK (ERK1/2 and p38)

• Regulation of cell cycle progression to create a favorable environment for viral replication

• Protection of infected cells from apoptosis, potentially extending the time available for viral replication

• Contribution to viral replication efficiency and plaque formation

Studies utilizing ORF12 deletion mutants (ROka12D) have demonstrated that while ORF12 is not absolutely essential for viral replication in cell culture, its absence results in reduced phosphorylation of ERK1/2 and Akt, smaller plaque sizes, and increased susceptibility to apoptosis .

Which cellular signaling pathways does VZV ORF12 protein activate?

VZV ORF12 protein activates multiple cellular signaling pathways that benefit viral replication:

• PI3K/Akt pathway: ORF12 protein binds to the p85 regulatory subunit of PI3K, leading to PI3K-dependent phosphorylation of Akt . This activation is time-dependent, with phosphorylation of Akt increasing at 12, 24, and 36 hours post-infection .

• MAPK pathways: ORF12 protein selectively triggers the phosphorylation of ERK1/2 (strongly) and p38 (partially), but not JNK . The activation of ERK1/2 by ORF12 is particularly robust, with experiments showing that ORF12 expression increases AP-1 reporter activity approximately 20-fold compared to control proteins .

• Cell cycle regulatory pathways: Through Akt activation, ORF12 influences the expression of cell cycle regulators, including increased levels of cyclin B1, cyclin D3, and phosphorylated glycogen synthase kinase 3β (GSK-3β) .

The pathway activation by ORF12 has been confirmed through multiple experimental approaches, including transfection studies with ORF12-expressing plasmids and infection studies comparing wild-type VZV with ORF12 deletion mutants .

How does ORF12 protein influence cell cycle progression?

VZV ORF12 protein regulates cell cycle progression through its activation of the PI3K/Akt pathway. Experimental evidence demonstrates several key effects:

• Alteration of cell phase distribution: Wild-type VZV infection reduces the G1 phase cell population and increases the M phase cell population. In contrast, infection with an ORF12 deletion mutant shows a reduced effect on G1 and M phase populations .

• Regulation of cell cycle proteins: VZV infection with wild-type virus increases levels of:

  • Cyclin B1 (M phase regulator)

  • Cyclin D3 (G1 phase regulator)

  • Phosphorylated glycogen synthase kinase 3β (GSK-3β)

• PI3K/Akt dependency: When Akt activity is inhibited with LY294002 (a PI3K inhibitor), the differences in cell cycle effects between wild-type and ORF12 mutant viruses are reduced, confirming that ORF12's cell cycle effects are mediated primarily through the PI3K/Akt pathway .

This cell cycle modulation likely creates a cellular environment that is more conducive to viral DNA replication. Since VZV can replicate in both dividing cells (like keratinocytes) and non-dividing cells (neurons), the ability of ORF12 to regulate the cell cycle may be particularly important for viral replication across diverse cell types in the body .

What is the molecular mechanism by which ORF12 activates the PI3K/Akt pathway?

The molecular mechanism of ORF12-mediated PI3K/Akt activation has been elucidated through detailed biochemical analyses. The process involves:

• Direct physical interaction: VZV ORF12 protein directly binds to the p85 regulatory subunit of PI3K. This interaction has been confirmed through co-immunoprecipitation experiments in infected cells. When ORF12 protein (tagged with V5) was immunoprecipitated, p85 was detected in the immune complex. Conversely, when p85 was immunoprecipitated, ORF12 protein was detected .

• Specificity of interaction: The interaction appears to be specific to p85, as ORF12 protein did not co-immunoprecipitate with Akt or ERK, indicating that the activation of Akt occurs through the canonical PI3K pathway rather than through direct interaction with Akt itself .

• PI3K-dependent Akt phosphorylation: Treatment of VZV-infected cells with the PI3K inhibitor LY294002 blocked Akt phosphorylation, demonstrating that ORF12-mediated Akt activation requires PI3K activity .

• Downstream effects: Once activated, the PI3K/Akt pathway leads to phosphorylation of GSK-3β, which influences cell cycle progression and survival. Infection with wild-type VZV induces stronger GSK-3β phosphorylation compared to infection with the ORF12 deletion mutant .

This mechanism appears to be somewhat unique among herpesviruses, as the HSV-1 ortholog of ORF12 (UL46) does not activate the AP-1 reporter in the same manner as VZV ORF12, suggesting functional divergence between these related viral proteins .

How does ORF12 protein differ from its orthologs in other herpesviruses?

VZV ORF12 protein exhibits functional differences from its orthologs in other herpesviruses, particularly HSV-1 VP11/12 (UL46):

• Signaling pathway activation: VZV ORF12 strongly activates the AP-1 reporter (approximately 20-fold compared to control), while HSV-1 UL46 does not activate this pathway at a significant level . This suggests a unique function of ORF12 in manipulating MAPK signaling.

• Cell survival function: While both VZV ORF12 and some herpesvirus orthologs contribute to protection against apoptosis, they do so through different mechanisms. ORF12 activates ERK and PI3K/Akt pathways, while HSV-2 ICP10 PK (another herpesvirus protein) also activates ERK but through its protein kinase domain .

• Structural implications: Although detailed structural comparisons aren't provided in the search results, the functional differences suggest structural or regulatory differences between ORF12 and its orthologs that account for their distinct signaling capabilities.

These differences highlight the evolutionary divergence among alpha-herpesviruses and suggest that each virus has evolved specific mechanisms to modulate host cell signaling to benefit viral replication and spread. Understanding these differences could provide insights into the pathogenesis of different herpesviruses and potentially reveal unique targets for antiviral therapies .

What are the consequences of ORF12 deletion on VZV replication and pathogenesis?

Deletion of ORF12 from the VZV genome results in several measurable effects on viral function:

• Reduced plaque size: VZV lacking ORF12 (ROka12D) forms smaller plaques compared to wild-type virus. This suggests that while ORF12 is not essential for viral replication, it contributes to the efficiency of viral spread .

• Altered signaling pathway activation: Cells infected with ORF12 deletion mutants show:

  • Reduced phosphorylation of ERK1/2

  • Decreased phosphorylation of Akt at 12, 24, and 36 hours post-infection

  • Lower levels of phosphorylated GSK-3β

  • Reduced levels of cyclin B1 and cyclin D3

• Increased susceptibility to apoptosis: Cells infected with ORF12 deletion mutants are more susceptible to staurosporine-induced apoptosis compared to wild-type VZV-infected cells, indicating that ORF12 contributes to the virus's ability to protect infected cells from apoptotic death .

• Cell cycle alterations: The deletion mutant has reduced ability to decrease G1 phase and increase M phase cell populations compared to wild-type virus, suggesting impaired ability to manipulate the host cell cycle .

These findings indicate that while ORF12 is not absolutely required for viral replication in cell culture, it plays important roles in optimizing the cellular environment for viral replication and spread. The protein's contribution to cell survival and cell cycle regulation likely enhances viral fitness in vivo, where the virus must overcome host immune responses and replicate in various cell types .

What experimental approaches can be used to study ORF12 protein interactions with cellular proteins?

Several effective experimental approaches have been employed to study ORF12 protein interactions with cellular proteins:

• Co-immunoprecipitation (Co-IP):

  • This technique was successfully used to demonstrate the interaction between ORF12 and the p85 subunit of PI3K .

  • For optimal results, cells were infected with a recombinant VZV expressing ORF12 protein with a V5 tag (ROka12DR) .

  • Immunoprecipitation was performed using antibodies to V5, p85, Flag, or Akt, followed by SDS-PAGE separation and immunoblotting with antibodies to p85 or V5 .

  • Controls included immunoprecipitation with irrelevant antibodies (Flag) and testing for non-interacting proteins (Akt, ERK) .

• Reporter assays:

  • AP-1 luciferase reporter assays were used to assess ORF12's ability to activate the AP-1 pathway .

  • This involved co-transfection of cells with (i) plasmids encoding ORF12, (ii) an AP-1 reporter plasmid expressing firefly luciferase under an AP-1-responsive promoter, and (iii) a control plasmid expressing Renilla luciferase .

  • Specificity was confirmed by testing against other reporters (NF-κB and ISRE) .

• Inhibitor studies:

  • Pathway-specific inhibitors (LY294002 for PI3K, U0126 for MEK1/2, SB202190 for p38, SP600125 for JNK) were used to determine which pathways were involved in ORF12-mediated effects .

  • These studies helped establish the PI3K-dependency of Akt activation by ORF12 .

• Comparative studies with viral mutants:

  • Creating ORF12 deletion mutants (ROka12D) and comparing their effects to wild-type virus provided powerful insights into ORF12 function .

  • This approach revealed ORF12's role in ERK1/2 and Akt phosphorylation, cell cycle regulation, and protection from apoptosis .

These methodologies collectively provide a robust framework for investigating viral protein interactions with host cellular components and their functional consequences.

What techniques are most effective for generating and validating VZV mutants with ORF12 modifications?

Several techniques have proven effective for generating and validating VZV mutants with ORF12 modifications:

• Cosmid-based mutagenesis system:

  • VZV mutants can be efficiently generated using a set of four overlapping cosmids that constitute the entire VZV genome .

  • For ORF12 deletion, researchers constructed cosmid NotI A12D with a deletion of all of ORF12 except the first 27 amino acids .

  • Transfection of melanoma (MeWo) cells with the modified cosmid along with the other three wild-type cosmids yielded the VZV deletion mutant (ROka12D) .

• Validation of mutants:

  • PCR verification: Primers flanking the ORF12 region can be used to confirm deletion, showing a smaller PCR product for the deletion mutant (0.3 kb) compared to wild-type virus (2.3 kb) .

  • Restriction enzyme analysis: BamHI digestion of virion DNA revealed characteristic pattern differences, with an 8.1-kb band in wild-type VZV and a 6.1-kb band in the ORF12 deletion mutant .

  • Southern blotting: Can be performed using a [32P]dCTP-labeled probe corresponding to VZV ORF12 to confirm the deletion .

• Revertant construction:

  • To ensure that phenotypic changes are specifically due to ORF12 deletion, creating a revertant virus is important.

  • This can be accomplished by inserting the ORF12 gene back into the deletion mutant, such as using the AvrII site in cosmid MstII .

• Functional validation:

  • Immunoblotting: To assess the impact on signaling pathways (phosphorylation of ERK1/2, Akt, GSK-3β) .

  • Plaque size measurements: To evaluate effects on viral spread .

  • Apoptosis assays: To determine the impact on cell survival .

  • Cell cycle analysis: To assess effects on cell cycle progression .

These techniques collectively provide a comprehensive approach to generating and characterizing VZV mutants, allowing researchers to determine the specific contributions of ORF12 to viral replication and pathogenesis.

What cell-based assays are most appropriate for studying ORF12-mediated effects on cell signaling?

Several cell-based assays have proven effective for studying ORF12-mediated effects on cell signaling:

• Phosphorylation assays via immunoblotting:

  • Western blotting using phospho-specific antibodies has been successfully used to detect activation of:

    • ERK1/2 (phosphorylated at Thr202/Tyr204)

    • Akt (phosphorylated at Ser473)

    • GSK-3β (phosphorylated at Ser9)

    • mTOR (phosphorylated at Ser2449)

  • This technique can be applied to both transfected cells expressing ORF12 and cells infected with wild-type versus ORF12 deletion mutant viruses .

• Luciferase reporter assays:

  • AP-1 luciferase reporter assays provide a quantitative measurement of MAPK pathway activation .

  • The dual-luciferase system using firefly luciferase under an AP-1-responsive promoter and Renilla luciferase as a transfection control enables accurate normalization .

  • Testing multiple reporter constructs (AP-1, NF-κB, ISRE) can confirm the specificity of ORF12's effects .

• Pathway inhibitor studies:

  • Treating cells with specific inhibitors helps delineate which pathways are involved:

    • LY294002 (PI3K inhibitor)

    • U0126 (MEK1/2 inhibitor)

    • SB202190 (p38 inhibitor)

    • SP600125 (JNK inhibitor)

  • These studies have revealed, for example, that ORF12-mediated AP-1 activation is strongly inhibited by MEK1/2 inhibitor, partially blocked by p38 inhibitor, but not affected by JNK inhibitor .

• Cell cycle analysis:

  • Flow cytometry analysis of DNA content can determine the distribution of cells in different cell cycle phases (G1, S, G2/M) .

  • This approach revealed that wild-type VZV infection reduces the G1 phase cell population and increases the M phase population, while the ORF12 deletion mutant has reduced effects .

• Apoptosis assays:

  • TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay can detect DNA fragmentation during apoptosis .

  • Cells can be treated with apoptosis inducers like staurosporine to assess the protective effects of ORF12 .

These assays provide complementary information about ORF12's effects on cell signaling, allowing researchers to build a comprehensive understanding of this viral protein's functions in modulating host cell pathways.

Comparative effects of wild-type VZV versus ORF12 deletion mutant on cellular signaling