Fig. 2: The proteins encoded by interferon-responsive genes are enriched in WT ORF1p-FLAG, but not ORF1p-FLAG (M8 [RBM]) mutant complexes.

a Experimental rationale for identifying host factors enriched in WT ORF1p-FLAG vs. ORF1p-FLAG (M8 [RBM]) immunoprecipitation reactions. Hypothetical diagrams of the proteins associating with WT and M8 (RBM) mutant RNP particles. Green circles, ORF1p-FLAG. Blue Oval, ORF2p. Red circle, purple squared oval, and green rectangle, host factors that might associate with ORF1p-FLAG and/or L1 RNPs. b The ORF1p (M8 [RBM]) mutant does not efficiently interact with Poly(A) Binding Protein Cytoplasmic 1 (PABPC1). HeLa-JVM cells were transfected with either pJM101/L1.3 (no FLAG), pJM101/L1.3FLAG (WT ORF1p-FLAG), or pALAF008 (ORF1p-FLAG [M8 [RBM]] mutant). An anti-FLAG antibody was used to immunoprecipitate ORF1p-FLAG. Western blots detected ORF1p (anti-FLAG), PABPC1 (anti-PABC1), and GAPDH (anti-GAPDH) in the input and IP fractions. GAPDH served as a sample processing control for the input fractions and a negative control in the IP experiments. c Separation of proteins associated with the WT and mutant ORF1p-FLAG proteins. The WT and M8 (RBM) mutant ORF1p-FLAG IP complexes were separated by SDS-PAGE using a 4-15% gradient gel and silver staining visualized the proteins. Protein size standards (kDa) are shown at the left of the gel. Black arrowhead, the expected molecular weight of ORF1p-FLAG. d Gene Ontology (GO) analysis identifies cellular proteins enriched in IP WT ORF1p-FLAG vs. the mutant ORF1p-FLAG complex. Cellular proteins present in the WT ORF1p and (M8 [RBM])-FLAG mutant IP complexes were identified using LC-MS/MS. Proteins having a >0.5 log₂ abundance ratio at any p-value in the WT ORF1p vs. M8 [RBM]) complexes were subjected to DAVID gene ontology analysis. Listed are the “functional annotation of UniProt Keyword GO biological process” terms. X-axis, protein count, the number of proteins identified by mass spectrometry that are included in each respective GO term. Y-axis, GO term. Circle size, −log₁₀FDR. Larger circles indicate higher confidence based on the FDR for each GO term. Red lettering, viral related GO terms. e GSEA preranked analysis identifies interferon-related gene sets enriched upon WT ORF1p-FLAG immunoprecipitation. Gene Set Enrichment Analysis (GSEA) of log₂ abundance ratio of cellular proteins immunoprecipitated in the WT ORF1p-FLAG vs. (M8 [RBM])-FLAG IP complexes was performed using hallmark gene sets in the Molecular Signatures Database (MSigDB: https://www.gsea-msigdb.org/gsea/msigdb/), followed by Leading Edge Analysis to determine gene set enrichment scores. The top six hallmark gene sets with the highest normalized enrichment score (NES) are sorted in descending values. X-axis, NES. Y-axis, hallmark gene sets. f The expression of engineered L1s modestly up-regulates IFN-α expression. HEK293T were transfected with either pCEP4 (an empty vector control), pJM101/L1.3FLAG (WT), pJM105/L1.3 (RT-), or pALAF008 (M8 [RBM]). RT-qPCR was used to quantify IFN-α (primer set: IFN-α, which amplifies IFN-α1 and IFN-α13) and L1 expression (primer set: mneoI [Alu or L1]) ~96 h post-transfection. IFN-α and L1 expression levels were normalized using β-actin (ACTB) as a control (primer set: Beta-actin). X-axis, name of constructs. Control, pCEP4. Y-axis, relative RNA expression levels normalized to the pCEP4 empty vector control. Red bars, normalized IFN-α expression levels. Gray bars, normalized L1 expression levels. Values from three independent biological replicates ± SEM are depicted in the graph. The p-values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests: pairwise comparisons of IFN-α relative to the pCEP4 control, p = 0.00028*** (WT); 0.00011*** (RT-); 3.14 × 10⁻⁶*** (M8 [RBM]). Pairwise comparisons of IFN-α: WT vs. RT-, p = 0.21^ns; WT vs. M8 (RBM), p = 0.00036***. Pairwise comparisons of L1 relative to WT, p = 0.87^ns (RT-), p = 0.10^ns (M8 [RBM]); ns: not significant; *** p < 0.001. For (d) and (e), the Source data are provided as a Source Data file.