Phosphorylation of shiftless is important for inhibiting the programmed −1 ribosomal frameshift

Fig. 1.. Biochemical characterization of recombinant human SFL. (A) Final purification of human SFL by size exclusion chromatography. Top inset: SDS-PAGE analysis of the peak fractions from the eluate. MW, molecular weight standards. (B) Analytical ultracentrifugation of the purified SFL protein. The calculated molecular mass is shown on top of the peak. (C) Top left: EMSAs for the interaction of SFL with different RNA substrates. −1 PRF-short is a 55-nt segment derived from the HIV-1 −1 PRF signal; its sequence and secondary structure are illustrated on the right, highlighted by red fonts. (CAA)20 is an ssRNA containing 20 copies of “CAA.” Representative of three independent experiments. (D) BLI titration of SFL against biotinylated −1 PRF-short RNA immobilized on SA biosensors. SFL concentration series used in the titration are labeled on the right. Binding kinetic parameters KD, kon, and koff are shown on top. Dashed lines are the fitting of the experimental data. Representative of two independent experiments.

Fig. 2.. Structural characterization of human SFL. (A) Schematics of the human SFL domain organization. SFL contains an N-terminal helical bundle domain (HB), three zinc fingers (ZF1 to ZF3), an HPL, and a C-terminal glutamate-rich domain (E-rich). (B) Ribbon model of human SFL with annotated secondary structures; α helices colored cyan, and β sheets colored magenta. Three zinc fingers are indicated, in which residues coordinating zinc ions are shown with stick models. Three boxes on the right are the magnified view of three zinc fingers with superimposed final 2Fo − Fc electron density maps (purple mesh, contour level 1.5 σ). HPL is colored red, on which phosphorylated S249, T250, T253, and S256 are shown with stick models. (C) Left: Bottom view of SFL (90° rotation about the horizontal axis with respect to the pose in B). Right: Stick model of HPL with superimposed final 2Fo − Fc electron density maps (purple mesh, contour level 1.5 σ). (D) Electrostatic potential surface plot of SFL; red to blue indicates negative to positive charge. Positively charged pockets 1 and 2 are identified on the SFL surface; residues constituting the positively charged pockets are shown with stick models and…

Fig. 3.. Ectopically expressed SFL is phosphorylated. (A and B) LC-MS/MS spectrum of the chymotrypsinized human SFL protein ectopically expressed in high-five insect cells, which identified four phosphorylated residues, pS249, pT250, pT253, and pS256. (C) Multiple sequence alignment of SFL homologs from humans and various animals, which shows that four phosphorylated residues S249, T250, T253, and S256 are invariant. (D and E) LC-MS/MS spectrum of the chymotrypsinized human SFL protein ectopically expressed in HEK293F mammalian cells, which identified four phosphorylated residues, pS249, pT250, pT253, and pS256.

Fig. 4.. Phosphorylation at HPL affects RNA binding. (A) EMSAs of the indicated SFL and SFL variants with the 55-nt −1 PRF-short RNA. Increasing protein concentrations used in EMSA are indicated on top of the gels. Representative of three independent experiments. (B) SDS-PAGE analysis of purified insect cell–expressed SFL, E. coli–expressed SFL, and insect cell–expressed SFL mutants S249A, T250A, T253A, S256A, S249D, T250D, T253D, and S256D. (C to G) BLI titrations of SFL mutants binding with −1 PRF-short RNA. Biotinylated −1 PRF-short RNAs were immobilized on SA biosensors for binding with SFL in solutions with a concentration series indicated on the top right. Binding kinetic parameters KD, kon, and koff are shown on top of each graph. Dashed lines are the fitting of the experimental data. Representative of two independent experiments. (C) Mutants S249A and S249D. (D) Mutants T250A and T250D. (E) Mutants T253A and T253D. (F) Mutants S256A and S256D. (G) Nonphosphorylated SFL expressed from E. coli. (H) Histogram illustrating the comparison of KD values of WT SFL and that of SFL mutants from the fitting of representative datasets. Error bars represent the fitting-derived KD error. (I)…

Fig. 5.. Phosphorylation at HPL affects SFL-mediated HIV-1 inhibition. (A and B) Western blot analysis of HIV-1 protein expression in HEK293T cells cotransfected with an HIV-1 reporter and constructs expressing SFL, SFLS, or indicated phosphomutants. [(A), top] Diagram of HIV-1 Gag-Pol genes and production of HIV-1 proteins Pr160 GagPol (dependent on −1 PRF), Pr55 Gag, and Pr24 CA (independent of −1 PRF). Western blot of HIV-1 protein expression, SFL and SFLS and β-actin in cells expressing the SFL mutants S249A, T250A, T253A, and S256A [(A), bottom] or mutants T250A, T250D, T253A, and T253D (B). Representative of three independent experiments. (C and D) Production of VSV-G (C) and HIV-1 Env (D) pseudotyped HIV-1 in HEK293T cells expressing SFL, SFLS, or indicated phosphomutants. Virus titers were determined by transduction in HeLa [(C), top], HEK293T [(C), bottom], MT4 [(D), top], and TZM-bl [(D), bottom] cells. n = 3 biological replicates. (E) Top: HIV-1 SG3 replication in Jurkat cells expressing SFL, SFLS, or phosphomutants. Virus growth was recorded from 72 to 168 hours postinfection. Bottom: Western blot analysis of the expression of Myc-SFL and mutants. n = 4 biological replicates.…

Fig. 6.. Phosphorylation is important to the SFL-mediated inhibition of −1 PRF. (A) Diagram of the dual-luciferase reporter systems harboring indicated −1 PRF signals (bottom graph and B). The −1 PRF sequence was inserted between the Rluc gene and the Fluc gene. Positive controls of the dual-luciferase reporter systems were prepared by adding a nucleotide (indicated by a red “N”) in the slippery sequence in the −1 PRF sequences; negative controls of the dual-luciferase reporter systems were prepared by adding a stop codon (indicated by a red “*”) in the slippery sequence of −1 PRF. Bottom: Control experiments of the dual-luciferase reporter systems validating the activity of different −1 PRF in the absence of SFL. CMV, cytomegalovirus. All data were shown as means ± SD. n = 3 biological replicates. (B) Eight reporters contained the −1 PRF sequences from HIV-1, HTLV-2, SARS-CoV-2, MERS-CoV, JEV, CHIKV, and PEG10 and CCR5 genes from humans. After transfection of the indicated reporter to HEK293T cells, luciferase activities were measured to calculate the Fluc/Rluc ratio that reflects the −1 PRF efficiency mediated by SFL, SFLS, and the phosphorylation mutants. EV, empty vector. All data…

Fig. 7.. Identification of kinases responsible for SFL phosphorylation. (A) Schematic diagram of three AirID constructs used for PDB. (B) Working flow of the proximity biotinylation for identifying kinases responsible for SFL phosphorylation. WB, Western blot. (C) The expression plasmids (A) were transfected into HEK293T cells, and 0.5 μM biotins were supplemented in the cell culture to promote biotinylation. The cells were harvested and lysed 16 hours posttransfection and analyzed by Western blot. Input: proteins before SA magnetic bead enrichment. Output: proteins after SA magnetic bead enrichment. Representative of three independent experiments. (D) LC-MS/MS analysis of SA magnetic bead–enriched samples from three independent biological replicates. Gray dots were proteins detected in both the 10- and 35-nm groups; black dot was SFL, and red dots were Ser/Thr kinases with expression levels at least fourfold higher than the AirID group (both 10 and 35 nm), among which six kinases (indicated with blue fonts) were selected for further analysis. FC, fold change. (E) Left: The purple and blue circles represent Ser/Thr kinases with expression levels up-regulated at least fourfold compared to…