Recombinant Walleye dermal sarcoma virus Protein ORF-C (orfC)

What is the genomic organization of WDSV and where is ORF-C located?

WDSV is a complex retrovirus in the Epsilonretrovirus genus that contains three accessory genes (orf a, orf b, and orf c) in addition to the standard retroviral gag, pol, and env genes. The orf c gene is specifically located between the 5' long terminal repeat (LTR) and the gag gene . This genomic positioning is significant as it allows ORF-C to be expressed from the full-length genomic transcript. The virus exhibits a seasonal cycle of tumor development and regression, with developing tumors containing low levels of orf a and orf b transcripts, while regressing tumors display high levels of genomic transcripts that encode ORF-C protein .

Experimental approaches to study the genomic organization typically involve PCR amplification of specific regions, sequence analysis, and characterization of transcripts using Northern blotting or RT-PCR. Understanding this organization is crucial for designing recombinant constructs and expression systems.

What is the subcellular localization of WDSV ORF-C and how can it be determined?

WDSV ORF-C exhibits a specific subcellular localization pattern critical to its function. When examined by immunofluorescence microscopy, ORF-C is observed throughout the cytoplasm but accumulates predominantly in cytoplasmic organelles, specifically targeting mitochondria . Dual-antibody staining for ORF-C and mitochondrial cytochrome c has demonstrated clear colocalization, indicating that ORF-C specifically targets these organelles .

To determine subcellular localization, researchers typically:

• Transfect cells with an expression vector encoding haemagglutinin-tagged ORF-C

• Perform immunofluorescence using anti-HA antibodies and mitochondrial markers

• Analyze using confocal microscopy to assess colocalization

• Confirm with biochemical fractionation and Western blotting

This mitochondrial localization contrasts with other WDSV accessory proteins, such as ORF-A (retroviral cyclin), which primarily localizes to the nucleus and interchromatin granule clusters where it interacts with transcription factors .

How does ORF-C induce apoptosis through mitochondrial disruption?

WDSV ORF-C induces apoptosis through direct targeting and disruption of mitochondrial function. Experimental evidence reveals several key mechanisms:

Cells transiently expressing ORF-C exhibit classic apoptotic morphology and lose mitochondrial membrane potential, as evidenced by their inability to retain MitoTracker Orange, a dye that specifically accumulates in active mitochondria . The colocalization with cytochrome c suggests that ORF-C may facilitate its release from mitochondria, a critical step in intrinsic apoptosis pathway activation . These findings strongly indicate that ORF-C's pro-apoptotic function contributes to tumor regression by inducing programmed cell death in infected cells.

What methods are used to detect and quantify ORF-C expression?

Researchers employ multiple complementary techniques to detect and quantify WDSV ORF-C expression:

• Immunological detection:

  • Western blot analysis using anti-ORF-C-specific antisera

  • Immunofluorescence with epitope-tagged constructs (typically HA-tagged)

  • Dual-staining to assess colocalization with cellular markers

• Transcript analysis:

  • RT-PCR to detect ORF-C-encoding transcripts

  • Northern blotting to quantify full-length genomic RNA

  • RNA sequencing to analyze expression patterns

• Recombinant expression systems:

  • Cell transfection with expression vectors

  • Generation of recombinant viruses (e.g., MYXVorfC)

  • Creation of stable cell lines with inducible expression

• Functional assays:

  • Mitochondrial activity using MitoTracker Orange retention

  • Apoptosis detection via phosphatidylserine exposure

  • Cytochrome c localization to detect mitochondrial permeabilization

For experimental design, it's important to note that ORF-C expression can be detected in tumor extracts with anti-ORF-C antisera, and its expression timing correlates with the regression phase of WDSV-associated tumors .

How does the seasonal cycle of WDSV infection relate to ORF-C expression?

WDSV exhibits a remarkable seasonal pattern of infection and gene expression that directly relates to ORF-C:

This seasonal pattern reveals a critical relationship between ORF-C expression and tumor regression . While developing tumors primarily express orf a and orf b transcripts, regressing tumors express approximately 1,000-fold higher levels of full-length genomic transcripts that encode ORF-C . This temporal pattern suggests that ORF-C plays a key role in tumor regression through its pro-apoptotic properties.

Experimental transmission studies have shown that only regressing spring tumor homogenates, which contain high levels of virus, can transmit the disease, while fall tumor homogenates cannot . This indicates that the switch to high-level virus expression, including ORF-C, is crucial for completing the viral life cycle.

What molecular mechanisms underlie ORF-C-induced mitochondrial dysfunction?

The molecular mechanisms of ORF-C-induced mitochondrial dysfunction involve several interconnected pathways:

• Mitochondrial targeting:

  • ORF-C specifically colocalizes with mitochondrial cytochrome c

  • This suggests interaction with outer mitochondrial membrane components

• Disruption of mitochondrial dynamics:

  • ORF-C expression leads to abnormal distribution of mitochondria in the cytoplasm

  • This may involve interference with mitochondrial fusion/fission machinery

• Membrane potential collapse:

  • Cells expressing ORF-C cannot retain MitoTracker Orange

  • This indicates disruption of the proton gradient essential for ATP production

• Apoptotic cascade initiation:

  • Increased surface phosphatidylserine exposure in ORF-C expressing cells

  • Release of pro-apoptotic factors from compromised mitochondria

Based on these observations, ORF-C likely disrupts mitochondrial function through either direct interaction with mitochondrial membrane components or by interfering with proteins that regulate mitochondrial integrity. The colocalization with cytochrome c suggests possible involvement in mitochondrial permeability transition, a key event in apoptosis initiation .

Advanced methodologies to further characterize these mechanisms would include mass spectrometry-based identification of ORF-C binding partners, structural studies of ORF-C's interaction with mitochondrial components, and live-cell imaging to track the dynamics of mitochondrial disruption in real-time.

How does recombinant expression of ORF-C affect virus pathogenicity?

Studies with recombinant myxoma virus expressing WDSV ORF-C (MYXVorfC) have provided valuable insights into how ORF-C affects virus pathogenicity:

This research demonstrates that MYXVorfC maintains its ability to replicate in permissive cells while showing attenuated pathogenicity in vivo . The recombinant virus was constructed by inserting the HA-tagged ORF-C gene between the M135 and M136 genes of MYXV, under the control of a late poxvirus promoter (vaccinia p11) . This strategic placement and promoter choice allowed complete virus replication before significant apoptosis was induced.

The attenuated pathogenicity of MYXVorfC, combined with its retained replicative capacity and enhanced apoptosis induction, makes it a promising candidate for oncolytic virus therapy development .

What is the relationship between ORF-C and other WDSV accessory proteins?

WDSV contains three accessory proteins with distinct functions and expression patterns:

ORF-A functions as a retroviral cyclin with limited homology to host cyclins . It localizes to the nucleus, interacts with components of the transcriptional co-activator complex (Mediator), and can regulate transcription via interaction with RNA polymerase II and cdk8 . ORF-A inhibits the WDSV promoter, suggesting it may suppress viral expression during tumor development .

In contrast, ORF-C targets mitochondria and induces apoptosis . This functional dichotomy suggests a coordinated role in the viral life cycle: ORF-A suppresses viral replication during tumor development, while ORF-C promotes cell death and virus release during tumor regression .

This relationship represents a sophisticated viral strategy to establish infection (tumor development) and then promote virus dissemination (tumor regression) in a seasonally regulated manner.

How can ORF-C be utilized in oncolytic virus design?

The pro-apoptotic properties of WDSV ORF-C make it a valuable component for enhancing oncolytic virus designs:

• Strategic incorporation:

  • ORF-C can be engineered into viruses like myxoma virus (as in MYXVorfC)

  • Expression should be controlled by late viral promoters to ensure virus replication precedes apoptosis

  • The vaccinia p11 promoter has been successfully used for this purpose

• Enhancing therapeutic efficacy:

  • ORF-C expression accelerates tumor cell death after virus replication

  • This may improve exposure of tumor antigens to the immune system

  • More rapid apoptosis could prevent antiviral responses from limiting virus spread

• Safety profile improvement:

  • Recombinant viruses expressing ORF-C show attenuated pathogenicity in vivo

  • MYXVorfC demonstrated delayed onset of clinical signs and extended survival in rabbits

  • This improved safety profile makes it attractive for clinical applications

• Reporter gene integration:

  • Combining ORF-C with reporter elements like tdTomato facilitates virus tracking

  • This approach was successful in the MYXVorfC design

• Potential immunotherapy synergy:

  • ORF-C-induced apoptosis may enhance tumor antigen release

  • This could potentially synergize with immune checkpoint inhibitors

Proof-of-concept studies with MYXVorfC demonstrated that the virus maintains replication competence in vitro while showing enhanced apoptosis induction and attenuated pathogenicity in vivo . These properties make ORF-C an attractive candidate for inclusion in next-generation oncolytic virus designs.

What challenges exist in studying ORF-C mechanisms across different cellular contexts?

Investigating ORF-C function across different cellular contexts presents several methodological and conceptual challenges:

• Species-specific considerations:

  • WDSV naturally infects walleye fish, but most laboratory studies use mammalian cells

  • Fish cells may respond differently to ORF-C than mammalian cells

  • Mitochondrial protein composition varies between species, potentially affecting ORF-C interactions

• Cell type-specific responses:

  • Tumor cells often have altered apoptotic pathways compared to normal cells

  • Different cell types express varying levels of Bcl-2 family proteins and other apoptosis regulators

  • The threshold for mitochondrial permeabilization differs between cell types

• Technical limitations:

  • Limited availability of fish-specific reagents for molecular studies

  • Challenges in establishing and maintaining fish cell lines

  • Limited cross-reactivity of antibodies between fish and mammalian proteins

• Contextual factors:

  • The tumor microenvironment differs substantially from in vitro conditions

  • Seasonal factors may influence ORF-C function in natural infections

  • Interactions with other viral proteins may modulate ORF-C activity

• Translational barriers:

  • Extrapolating from fish viral proteins to mammalian therapeutic applications

  • Optimizing ORF-C expression levels for therapeutic benefit

  • Addressing potential immunogenicity in mammalian systems

Addressing these challenges requires multidisciplinary approaches including comparative studies across species and cell types, development of fish-specific molecular tools, advanced imaging techniques, and structure-function analyses to identify key domains for apoptosis induction. Overcoming these challenges will be essential for harnessing the full therapeutic potential of ORF-C in cancer treatment strategies.