Recombinant Xenopus laevis UPF0515 protein C19orf66 homolog

What is the Xenopus laevis UPF0515 protein C19orf66 homolog and how is it classified?

The Xenopus laevis UPF0515 protein C19orf66 homolog, also known as SHFL.L (shiftless antiviral inhibitor of ribosomal frameshifting L homeolog), is an interferon-stimulated gene (ISG) product with significant antiviral properties. It has several synonyms including c19orf66.L, repressor of yield of DENV protein homolog, and shiftless antiviral inhibitor of ribosomal frameshifting protein homolog. Its NCBI Gene ID is 444483 in Xenopus laevis . The protein belongs to a family of viral restriction factors that interfere with viral replication through multiple mechanisms, most notably by inhibiting programmed ribosomal frameshifting.

What are the known antiviral functions of C19orf66 homologs across species?

C19orf66 homologs have been demonstrated to exhibit broad antiviral activity against multiple virus families. In humans, C19orf66 (also annotated as RyDEN, IRAV, and SVA-1) has been shown to restrict dengue virus (DENV) replication by interacting with cytoplasmic poly(A) binding protein (PABPC) and the RNA helicase MOV10 . It also demonstrates significant activity against Japanese encephalitis virus (JEV) by inhibiting frameshift-mediated expression of NS1' and promoting degradation of the JEV NS3 protein through the lysosome pathway . Additionally, research has shown it restricts Kaposi's sarcoma-associated herpesvirus (KSHV) infection by impeding early viral gene expression . The Xenopus laevis homolog is presumed to have similar functions, though species-specific variations may exist.

How does the expression pattern of C19orf66 change during viral infection?

During viral infection, C19orf66 expression is significantly upregulated as part of the interferon response. Studies specifically examining KSHV infection showed that C19orf66 expression increases upon viral lytic reactivation and continues to accumulate over the course of 96 hours post-reactivation . This upregulation pattern is consistent with its role as an interferon-stimulated gene. Importantly, while many host transcripts are degraded by viral endonucleases during infection, C19orf66 transcripts systematically escape this degradation, suggesting an evolutionary adaptation that allows this antiviral factor to maintain its protective effects even in the presence of viral host-shutoff mechanisms .

Through what mechanisms does C19orf66 inhibit viral replication?

C19orf66 employs multiple mechanisms to inhibit viral replication, which vary somewhat depending on the virus:

• Inhibition of Programmed Ribosomal Frameshifting (-1 PRF): C19orf66 targets the -1 programmed ribosomal frameshifting mechanism used by viruses like Japanese encephalitis virus (JEV), preventing the expression of certain viral proteins such as NS1' .

• Protein Degradation: It promotes the degradation of viral proteins through the lysosome pathway, as demonstrated with JEV NS3 protein .

• Restriction of Early Viral Gene Expression: In KSHV infection, C19orf66 restricts the expression of early and delayed early viral genes, resulting in lower levels of viral reactivation and reduced yield of infectious viral particles .

• RNA Processing Interference: Research suggests it may influence viral RNA stability and processing by interacting with host RNA-binding proteins such as PABPC and MOV10 .

What structural elements of C19orf66 are responsible for its escape from virus-induced RNA degradation?

The 3' untranslated region (3' UTR) of C19orf66 contains structural elements that protect it from degradation by viral endonucleases. These elements, similar to the previously identified SOX resistance elements (SREs), enable C19orf66 transcripts to escape degradation by multiple herpesviral endonucleases, including SOX (from KSHV), muSOX (from MHV68), BGLF5 (from EBV), and vhs (from HSV-1) . This protection mechanism allows C19orf66 to continue expressing during infection when most host transcripts are degraded, enabling sustained antiviral activity. The specific sequence features of these protective elements have not been fully characterized but appear to function across diverse viral endonucleases .

How can researchers experimentally measure the antiviral activity of recombinant C19orf66?

Researchers can measure the antiviral activity of recombinant C19orf66 through several experimental approaches:

• Viral Replication Assays: Overexpression or knockdown of C19orf66 followed by infection with target viruses and quantification of viral load using qPCR, plaque assays, or TCID50 assays.

• Reporter Gene Assays: Using viruses expressing reporter genes (such as GFP or RFP) to visualize and quantify infection levels. For example, with KSHV.219 virus that expresses GFP constitutively and RFP under lytic promoter control .

• Supernatant Transfer Assays: Collecting supernatants from infected cells with or without C19orf66 manipulation and using them to infect new cells to quantify infectious viral particle production .

• Viral Gene Expression Analysis: RT-qPCR to measure expression levels of viral genes (early, delayed early, and late) in the presence or absence of C19orf66 .

• Protein-Protein Interaction Studies: Co-immunoprecipitation or proximity ligation assays to identify interactions between C19orf66 and viral or host proteins.

What expression systems are most effective for producing recombinant Xenopus laevis C19orf66 homolog?

While the search results don't specifically address expression systems for the Xenopus laevis C19orf66 homolog, based on related research with C19orf66 from other species, effective expression systems would include:

• Bacterial Expression Systems: E. coli-based systems (such as BL21(DE3)) with appropriate codon optimization for Xenopus laevis sequences. These systems are advantageous for high yield but may require careful optimization for proper folding of eukaryotic proteins.

• Insect Cell Expression: Baculovirus expression systems provide eukaryotic post-translational modifications and often yield properly folded proteins.

• Mammalian Expression Systems: HEK293T or similar cell lines transfected with expression vectors containing the Xenopus laevis C19orf66 homolog gene can produce functional protein with appropriate modifications.

• Cell-Free Expression Systems: These may be useful for rapid screening of constructs and variants.

The choice would depend on the specific research questions, required protein modifications, and downstream applications.

What are the key considerations for designing experiments to study C19orf66 function across different viral infection models?

When designing experiments to study C19orf66 function across different viral infection models, researchers should consider:

• Cell Type Selection: Choose cell lines that support both C19orf66 expression and viral replication. For studying the Xenopus laevis homolog, consider using amphibian cell lines or testing cross-species functionality in mammalian cells.

• Expression Level Control: Use inducible expression systems to control protein levels, as overexpression may cause artifacts.

• Knockdown/Knockout Strategies: Design targeted siRNAs or CRISPR/Cas9 guides specific to the Xenopus laevis sequence to assess loss-of-function effects.

• Viral Diversity: Test multiple virus families to determine specificity or breadth of antiviral activity, as C19orf66 has shown effects against flaviviruses (JEV, DENV) and herpesviruses (KSHV) .

• Domain Analysis: Create deletion or point mutation constructs to map functional domains, particularly those involved in frameshift inhibition or protein degradation mechanisms.

• Temporal Dynamics: Monitor both C19orf66 expression and viral replication over time, as studies with KSHV showed increased accumulation over 96 hours post-reactivation .

• Subcellular Localization: Track protein localization during infection, although studies with KSHV did not find differential shuttling upon viral reactivation .

How conserved is C19orf66 across species, and what does this suggest about its evolutionary importance?

While the search results don't provide comprehensive information about C19orf66 conservation across all species, they do mention that C19orf66 orthologs were identified in 11 amphibian species in a study related to fertilization mechanisms in Xenopus laevis . The fact that C19orf66 functions as an antiviral factor against diverse viruses including flaviviruses (JEV, DENV) and herpesviruses (KSHV) suggests evolutionary conservation of its antiviral function .

The ability of C19orf66 transcripts to escape degradation by various viral endonucleases through elements in their 3' UTR further suggests an evolutionary adaptation that maintains antiviral defense even during active viral infection . This escape mechanism appears to work against endonucleases from phylogenetically diverse herpesviruses (KSHV, MHV68, EBV, HSV-1), indicating broad evolutionary conservation of this protective feature .

How do the functional domains of Xenopus laevis C19orf66 homolog compare to human C19orf66?

Based on the search results, we can infer some functional comparisons between Xenopus laevis C19orf66 homolog and human C19orf66:

• Zinc-Finger Domain: Studies of human C19orf66 have identified a zinc-finger domain that is important for certain antiviral functions. For instance, C19orf66-Zincmut (with mutations in this domain) shows differential restriction capabilities against Hepatitis C virus compared to other variants . The search results don't specifically describe this domain in the Xenopus laevis homolog, but it likely has similar functional importance if conserved.

• Frameshifting Inhibition: Both human C19orf66 and presumably its Xenopus homolog can inhibit programmed -1 ribosomal frameshifting, a key mechanism for viral protein expression in many viruses .

• Protein Interaction Domains: Human C19orf66 interacts with cytoplasmic poly(A) binding protein (PABPC) and RNA helicase MOV10 to restrict dengue virus replication . The Xenopus homolog likely has similar interaction domains, though species-specific differences may exist.

Without more specific data on the Xenopus laevis homolog's structure, a complete domain-by-domain comparison is not possible from the search results alone.

How might understanding the mechanism of C19orf66 escape from viral endonucleases contribute to novel antiviral therapeutics?

Understanding how C19orf66 escapes viral endonuclease-mediated RNA degradation could contribute to novel antiviral therapeutics in several ways:

• Protective RNA Elements: The SOX resistance elements (SREs) or SRE-like elements in the 3' UTR of C19orf66 that protect it from viral endonucleases could be identified and incorporated into mRNA-based therapeutics or vaccines to increase stability during viral infection .

• Broad-Spectrum Antiviral Strategies: Since C19orf66 transcripts escape degradation by endonucleases from diverse herpesviruses (KSHV, MHV68, EBV, HSV-1), understanding this mechanism could lead to broad-spectrum antiviral approaches .

• Enhanced Antiviral Protein Expression: Incorporating these protective elements into expression constructs for other antiviral proteins could enhance their expression during active viral infection when host shutoff normally occurs.

• Novel Drug Targets: Identifying the molecular interactions that protect certain transcripts from viral endonucleases could reveal new druggable targets to counteract viral host shutoff mechanisms.

• Combination Approaches: Since C19ORF66 restricts viral replication through multiple mechanisms (frameshifting inhibition, protein degradation, early gene expression), a comprehensive understanding could inform combination therapeutic approaches targeting multiple viral life cycle stages .

What are the key research gaps in understanding the full antiviral spectrum of C19orf66 homologs?

Several important research gaps exist in understanding the full antiviral spectrum of C19orf66 homologs:

• Species-Specific Variations: While human C19orf66 has been studied against multiple viruses, the specific antiviral activities of the Xenopus laevis homolog remain largely unexplored. Comparative functional studies would reveal whether antiviral mechanisms are conserved across species.

• Comprehensive Viral Range: Current research has demonstrated C19orf66 activity against flaviviruses (JEV, DENV) and herpesviruses (KSHV) , but its activity against other virus families requires investigation.

• Structure-Function Relationships: Detailed structural studies of C19orf66 would help elucidate how specific domains contribute to its diverse antiviral functions.

• Regulation Mechanisms: While C19orf66 is known to be interferon-stimulated, the complete regulatory network controlling its expression in different tissues and infection scenarios is not fully characterized.

• Interaction Networks: Comprehensive identification of C19orf66 protein-protein and protein-RNA interactions would provide deeper insight into its mechanisms of action.

• Viral Evasion Strategies: Research into whether viruses have evolved specific mechanisms to counteract C19orf66 restriction would advance understanding of virus-host co-evolution.

• Tissue-Specific Functions: Investigation of whether C19orf66 functions differently in various tissues or developmental stages, especially in Xenopus laevis, would provide valuable insights into its biological roles.

How can structural biology approaches be applied to understand the mechanism of C19orf66-mediated inhibition of programmed ribosomal frameshifting?

Structural biology approaches can significantly advance understanding of C19orf66-mediated inhibition of programmed ribosomal frameshifting through:

These approaches, combined with functional assays measuring frameshifting efficiency (such as dual-luciferase reporter systems), would provide mechanistic insights into how C19orf66 specifically targets viral -1 PRF while allowing normal translation to proceed .

What are the most significant challenges in purifying functional recombinant Xenopus laevis C19orf66 homolog for structural studies?

Major challenges in purifying functional recombinant Xenopus laevis C19orf66 homolog for structural studies include:

• Protein Solubility: Ensuring the recombinant protein remains soluble throughout the purification process, potentially requiring optimization of buffer conditions, salt concentrations, and pH.

• Post-translational Modifications: If the Xenopus laevis C19orf66 homolog requires specific post-translational modifications for functionality, appropriate expression systems (eukaryotic rather than prokaryotic) would be necessary.

• Structural Integrity: Maintaining the native conformation of the protein, particularly if it contains zinc-finger domains similar to human C19orf66, which could require specific metal ion concentrations in buffers.

• Protein-Protein Interactions: If the protein functions as part of a complex, co-expression with binding partners might be necessary to maintain stability and function.

• RNA Binding Properties: Since C19orf66 likely interacts with RNA, preventing non-specific RNA binding during purification may present challenges.

• Expression Levels: Optimizing expression conditions to achieve sufficient yield for structural studies without compromising protein quality.

• Crystallization Barriers: For X-ray crystallography, obtaining diffraction-quality crystals can be particularly challenging for RNA-binding proteins with flexible domains.

How can researchers effectively differentiate between direct and indirect antiviral effects of C19orf66 in experimental settings?

To effectively differentiate between direct and indirect antiviral effects of C19orf66, researchers should consider the following approaches:

• In Vitro Reconstitution Assays: Using purified components to test direct effects on viral processes such as frameshifting or protein stability, isolated from cellular contexts.

• Structure-Function Analysis: Creating point mutations or domain deletions to map which regions of C19orf66 are essential for different antiviral activities.

• Temporal Analysis: Examining the kinetics of C19orf66 effects on viral replication to distinguish immediate (likely direct) from delayed (potentially indirect) effects.

• Cell-Free Systems: Testing C19orf66 effects on viral RNA translation or replication in cell-free extracts to eliminate cellular signaling components.

• Binding Studies: Direct measurement of interactions between C19orf66 and viral components using techniques such as surface plasmon resonance (SPR), microscale thermophoresis (MST), or bio-layer interferometry (BLI).

• Interferon-Blocking Experiments: Using JAK/STAT inhibitors or cells deficient in interferon signaling to determine whether observed antiviral effects depend on secondary interferon responses.

• Comparative Viral Susceptibility: Testing C19orf66 effects against viruses with known differences in specific processes (e.g., those that use frameshifting versus those that don't) to identify mechanism-specific patterns of inhibition.

For example, research has shown that C19orf66 inhibits JEV replication by both hindering frameshift-mediated expression of NS1' and promoting degradation of the JEV NS3 protein through the lysosome pathway . These distinct mechanisms were identified through careful experimental design and controls.