Recombinant Mouse UPF0515 protein C19orf66 homolog

What is the C19orf66 protein and its mouse homolog?

C19orf66 is a novel interferon-stimulated gene product that exhibits significant activity against various viral infections, particularly flaviviruses. The mouse homolog shares functional characteristics with the human version, demonstrating antiviral properties. Research has shown that C19orf66 can inhibit Japanese encephalitis virus (JEV) replication through multiple mechanisms, including targeting programmed -1 ribosomal frameshifting (-1 PRF) and promoting the degradation of viral NS3 protein via the lysosome-dependent pathway . When studying this protein, researchers typically use recombinantly expressed versions that enable controlled experimental conditions.

What expression systems are recommended for producing recombinant mouse C19orf66?

While specific information about mouse C19orf66 production is limited in the search results, typical approaches for recombinant mouse proteins involve bacterial expression systems such as E. coli, similar to other recombinant mouse proteins like GDF-15 . For optimal purification and downstream applications, consider adding affinity tags (such as a C-terminal His-tag) to facilitate isolation. The expression system should be selected based on the intended application, with bacterial systems providing higher yields but mammalian systems potentially offering better folding and post-translational modifications.

What are the optimal storage and handling conditions for recombinant mouse C19orf66?

Based on protocols for similar recombinant proteins, the following guidelines are recommended:

• Store lyophilized protein in a manual defrost freezer at -20°C to -80°C

• Avoid repeated freeze-thaw cycles that can compromise protein integrity

• Reconstitute using sterile conditions in an appropriate buffer (typically PBS or a low concentration of HCl, depending on protein properties)

• After reconstitution, aliquot the protein solution to minimize freeze-thaw cycles

• For long-term storage of reconstituted protein, store at -80°C

What functional assays are most effective for evaluating mouse C19orf66 antiviral activity?

When evaluating the antiviral activity of mouse C19orf66, consider the following experimental approaches:

• Viral replication assays: Overexpression of C19orf66 in appropriate cell lines (such as 293T cells) followed by viral challenge and quantification of viral RNA or protein levels.

• Knockdown experiments: siRNA-mediated depletion of endogenous C19orf66 in cells like HeLa or A549, which has been shown to significantly increase virus replication, confirming the protein's antiviral role .

• Frameshift efficiency assays: Since C19orf66 has been shown to inhibit the frameshift production of JEV NS1′, assays measuring the NS1′/NS1 ratio can directly evaluate this mechanism of action .

• Protein degradation assays: To assess the effect on viral NS3 protein degradation, researchers should include lysosomal inhibitors to confirm the pathway involved in C19orf66-mediated protein degradation .

How can researchers distinguish between the different antiviral mechanisms of C19orf66?

C19orf66 appears to employ at least two distinct mechanisms for inhibiting viral replication:

• Targeting -1 PRF: To isolate this mechanism, design experiments using reporter constructs containing the viral frameshift signal without producing complete viral proteins. This allows assessment of frameshifting efficiency without confounding effects from other mechanisms.

• NS3 degradation pathway: To specifically study this mechanism, use constructs expressing NS3 protein alone and measure its stability in the presence or absence of C19orf66. Include lysosomal inhibitors (e.g., chloroquine) to confirm the degradation pathway.

• Mutant controls: Include C19orf66 mutants in experiments as functional controls. Both C19orf66-209 and C19orf66-Zinc mut have demonstrated weaker antiviral effects than wild-type C19orf66 and do not significantly affect the NS1′/NS1 ratio or NS3 expression, making them valuable experimental controls .

What controls should be included when studying recombinant mouse C19orf66?

Comprehensive experiments should include:

• Negative controls:

  • Empty vector controls for overexpression studies

  • Non-targeting siRNA for knockdown experiments

  • Unrelated proteins of similar size/structure to control for non-specific effects

• Functional mutant controls:

  • C19orf66-209 variant (truncated form)

  • C19orf66-Zinc mut (mutation in the zinc-binding domain)

  • These mutants have demonstrated reduced antiviral activity and can help identify domain-specific functions

• Positive controls:

  • Known antiviral ISGs with similar mechanisms

  • Human C19orf66 for comparative studies

How does C19orf66 inhibit viral programmed -1 ribosomal frameshifting?

C19orf66 has demonstrated a specific inhibitory effect on the frameshift production of JEV NS1′, which relies on -1 PRF. This inhibition is enhanced when C19orf66 and JEV NS1-NS2A are co-expressed in cells . The mechanism appears to be domain-specific, as mutant forms C19orf66-209 and C19orf66-Zinc mut did not significantly alter the NS1′/NS1 ratio.

Potential mechanisms include:

• Direct interaction with RNA structures that facilitate frameshifting

• Interaction with ribosomal components required for -1 PRF

• Recruitment of host factors that interfere with the frameshifting process

To investigate this mechanism further, researchers should:

• Perform in vitro translation assays with purified recombinant C19orf66

• Conduct RNA binding assays to assess potential direct interactions

• Use structural approaches to characterize protein-RNA or protein-ribosome interactions

What is the role of the zinc-binding domain in C19orf66's antiviral function?

The zinc-binding domain appears crucial for C19orf66's full antiviral activity. Research has shown that C19orf66-Zinc mut has weaker antiviral effects compared to wild-type C19orf66 . This domain likely plays important roles in:

• Protein structure stabilization

• RNA recognition and binding

• Protein-protein interactions with host or viral factors

Researchers investigating this domain should consider:

• Structural studies comparing wild-type and zinc mutant proteins

• Protein interaction analyses to identify binding partners affected by zinc domain mutations

• Comparative studies between human and mouse homologs to identify conserved functional elements

How does C19orf66 promote the lysosomal degradation of viral NS3 protein?

C19orf66 has been shown to down-regulate JEV NS3 protein expression via the lysosome-dependent pathway . This represents a distinct antiviral mechanism from its effect on -1 PRF.

To characterize this mechanism, researchers should:

• Determine whether C19orf66 directly interacts with NS3 through co-immunoprecipitation or proximity labeling

• Investigate whether C19orf66 recruits components of the lysosomal targeting machinery to NS3

• Examine whether post-translational modifications of NS3 are involved in targeting it for degradation

• Test whether this mechanism extends to NS3 proteins from related flaviviruses

How should researchers interpret differences between human and mouse C19orf66 activity?

When comparing human and mouse C19orf66, consider:

• Evolutionary conservation: Analyze sequence homology to identify conserved domains likely essential for core functions versus divergent regions that may confer species-specific activities.

• Viral substrate specificity: Test both homologs against a panel of viruses to determine whether their antiviral spectra differ. C19orf66 has shown activity against multiple viruses, including JEV and Hepatitis C virus .

• Mechanistic differences: Assess whether both homologs employ the same antiviral mechanisms (PRF inhibition and NS3 degradation) with similar efficiency or if species-specific mechanisms exist.

A comprehensive analysis should include:

• Sequence and structural comparisons

• Functional assays against multiple viral targets

• Domain swapping experiments to identify regions responsible for any observed differences

What factors might affect the reproducibility of C19orf66 experiments?

Several factors can impact the reproducibility of C19orf66 experiments:

• Protein quality and stability:

  • Ensure proper storage conditions for recombinant protein

  • Validate protein activity before experiments

  • Consider carrier-free preparations for certain applications to avoid interference

• Expression levels:

  • Standardize expression levels across experiments

  • Consider both transient and stable expression systems

  • Verify expression using appropriate detection methods

• Cell type variations:

  • Different cell types may have varying levels of endogenous C19orf66

  • Cell type-specific cofactors may influence activity

  • Interferon signaling status of cells might affect results

• Viral strain differences:

  • Use well-characterized viral strains

  • Consider potential strain-specific resistance mechanisms

Table 5.1: Functional Comparison of Wild-type and Mutant C19orf66

This table summarizes the differential effects of wild-type and mutant forms of C19orf66 on various aspects of JEV infection based on current research findings .

How does C19orf66 compare to other interferon-stimulated genes with antiviral activity?

C19orf66 represents a novel ISG with specific mechanisms targeting viral translation processes and protein stability. Compared to other ISGs:

• Unique mechanisms: C19orf66's ability to target -1 PRF represents a relatively uncommon antiviral mechanism among ISGs. This provides potential advantages for targeting viruses that rely on this translation mechanism.

• Multiple modes of action: The combination of targeting both -1 PRF and promoting NS3 degradation suggests C19orf66 employs multiple antiviral strategies, which may reduce the likelihood of viral escape.

• Potential synergies: When designing experiments to study C19orf66 in the context of the broader interferon response, researchers should consider potential synergistic interactions with other ISGs that target different stages of the viral lifecycle.

What are the most promising applications for recombinant mouse C19orf66 in antiviral research?

Based on current findings, promising research directions include:

• Broad-spectrum antiviral development: C19orf66's ability to target -1 PRF, which is utilized by various viruses including flaviviruses and coronaviruses, suggests potential for broad-spectrum antiviral applications .

• Structure-based drug design: Elucidating the structural basis of C19orf66's interaction with the frameshift elements could enable the development of small molecules that mimic this activity.

• Combination therapies: Investigating potential synergies between C19orf66 and other antiviral agents targeting different viral processes.

• Animal models: Studying the role of mouse C19orf66 in viral pathogenesis using knockout or transgenic mouse models.

How might structural studies advance our understanding of C19orf66 function?

Structural characterization of C19orf66 would significantly advance our understanding of its mechanisms by:

• Identifying key domains and residues involved in:

  • RNA binding and PRF inhibition

  • Protein-protein interactions leading to NS3 degradation

  • Zinc coordination and its structural importance

• Revealing conformational changes that might occur upon:

  • Binding to viral RNA or proteins

  • Interaction with host factors

  • Interferon-induced post-translational modifications

• Guiding the development of:

  • Improved mutants for experimental studies

  • Structure-based inhibitors or mimetics

  • Engineered versions with enhanced antiviral properties