Article Figures & Data
Figures
- FIG 1
The pandemic 2009 H1N1 and H1N1 viruses from the 1930s are resistant to TRIM22 restriction. (A) Control and human TRIM22-transduced MDCK cells were infected with H1N1 strains at an MOI of 0.001. Viral titers were measured in supernatants harvested 24, 48, and 72 hpi by plaque assay, indicated as plaque-forming units (PFU). As a positive control for TRIM22 restriction activity, A/New Caledonia/20/99 (sH1N1) and two additional seasonal H1N1 strains, A/USSR/90/77 and A/Paris/1149/2008, were used to infect TRIM22-overexpressing MDCK cells. Viral titers are expressed as the means ± standard errors of the means (SEM) from three experiments performed in duplicate. P values were determined using two-way ANOVA (*, P < 0.05; ***, P < 0.001). (B) Total cell extracts from control and TRIM22-overexpressing MDCK cells infected with different H1N1 strains were analyzed by Western blotting, and viral NP expression levels were evaluated. TRIM22 expression was also examined, and β-actin was used as a normalizer. These results are representative of one out of three independent experiments.
- FIG 2
pH1N1 NP is more resistant to TRIM22-mediated inhibition than sH1N1 NP in a polymerase activity assay. Expression plasmids for the polymerase subunits PB2, PB1, and PA from the pH1N1 virus and NP expression plasmids from either the pH1N1 or sH1N1 strains were cotransfected with a plasmid carrying a pseudoviral genome, harboring a Renilla reporter, into HEK293T cells either without (Nil) or with increasing concentrations of TRIM22-expressing plasmid. The reporter gene activity was measured 24 h later. (A) Increasing amounts of exogenous beta interferon (IFN-β) were added to the polymerase activity assay. The sH1N1 NP-dependent polymerase activity was more sensitivity to the IFN-β stimulation than that of pH1N1 NP. Means ± standard deviations (SD) from 3 independent experiments in triplicates are reported. P values were determined using one-way ANOVA with Bonferroni’s multiple-comparison test of Nil versus each treatment (****, P < 0.0001). (B) Forty nanograms of TRIM22-expressing plasmid reduced the polymerase activity driven by the sH1N1 NP by 50% but not the polymerase activity driven by the pH1N1 NP. Means ± SEM from 3 independent experiments in triplicate are reported. P values were determined using one-way ANOVA with the Bonferroni’s multiple-comparison test of nil versus each treatment (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Western blot analysis was performed to determine the expression levels of TRIM22 and both pH1N1 and sH1N1 NPs.
- FIG 3
NP sequence alignment of human H1N1 strains and phylogenetic analysis of representative NP from pandemic and seasonal H1N1 strains. (A) The sequence comparison of NP from H1N1 strains (A/Brevig Mission/1918, A/WSN/33, A/PR/8/34, A/USSR/90/77, A/New Caledonia/20/99, A/Brisbane/59/2007, A/Paris/1149/2008 and A/Paris/2590/2009) shows 15 conserved lysine residues (in green). In A/New Caledonia/20/1999 and A/Brisbane/59/2007 isolates, arginine residues have been substituted for by lysines (in blue) at four specific sites (K98, K293, K422, and K446). Four arginine residues (R98, R293, R422, and R446) are present in the pandemic A/Paris/2590/2009 strain. (B) The maximum likelihood tree of 12 selected sequences shows two major branches that separate the H1N1 strains derived from the 1918 Brevig H1N1 strain from those derived from the swine 2009 pandemic H1N1 strain. Numbers at the nodes indicate bootstrap values obtained after 1,000 replications. A red asterisk indicates a viral isolate with four lysine residues, whereas a black asterisk indicates a viral isolate with four arginine residues. (C) Three major pandemics occurred during the 20th century, including the “Spanish” influenza pandemic (H1N1, 1918), the “Asian” pandemic (H2N2, 1957), and the “Hong Kong” pandemic (H3N2, 1968). H1N1 reemerged in 1977 and continued to circulate in the human population until 2009, when the pandemic H1N1 strain emerged. The continuity of the NP lineage along H1N1-H2N2-H3N2 viruses and the R-to-K changes are shown.
- FIG 4
Localization of TRIM22-sensitive lysine residues in the NP molecular structure. Shown is a structural model of the mutant form of NP resistant to TRIM22 restriction. (A) The model of NPR4/K4, an NP with 4 R-to-K single-point mutations (R98K, R293K, R422K, and R446K), was generated using PDB code 2IQH as the template (18). NP is represented as cartoons, with the head and the body of the protein colored in pink and cyan, respectively. K98, K293, K422, and K446 are highlighted as sticks and numbered with the single-letter code. (B) The interactions predicted for K98 (left), K293 (middle), and K422 and K446 (right) are shown. Residues are highlighted as sticks and numbered with the single-letter code. (C) The model of NPR4/K4 in a helical ribonucleoprotein-like structure using PDB code 4BBL as the template (21) is shown as the surface. Images were prepared using pymol-v1.8.4.2 (PyMOL Molecular Graphics System, version 1.8; Schrödinger, LLC) and the Maestro software package (42).
- FIG 5
Arginine-to-lysine mutations conferring TRIM22 restriction and lysine-to-arginine mutations causing loss of TRIM22 restriction. TRIM22-mediated restriction activity was tested in the polymerase activity assay in the presence of 40 ng of TRIM22-expressing plasmid. (A) The bars show the activity of WT pH1N1 NP and R-to-K mutants relative to the same samples tested in the absence of TRIM22-expressing plasmid, which was set to 100%. Means ± SD from three independent experiments performed in triplicate are reported. The results of one-way ANOVA with the Bonferroni’s multiple-comparison test of WT versus each mutant are shown (*, P < 0.05; ****, P < 0.0001; ns, not significant). The expression levels of pH1N1 NP and TRIM22 were determined by Western blotting of WCE obtained by pooling the triplicate wells of each experiment. One representative Western blot of three is shown. (B) The bars show the activity of sH1N1 NP WT and K-to-R mutants relative to the same samples tested in the absence of TRIM22-expressing plasmid, which was set to 100%. Means ± SD from three independent experiments performed in triplicate are reported. The results of one-way ANOVA with the Bonferroni’s multiple-comparison test of WT versus each mutant are shown (****, P < 0.0001). Western blot analysis was performed to determine the expression levels of sH1N1 NP and TRIM22. (C) Quantification of the band intensity was performed by using ImageJ. Means ± SEM of the ratio between the intensity of NP bands and β-actin are reported for 3 independent experiments. The results of one-way ANOVA with the Bonferroni’s multiple-comparison test are shown (*, P < 0.05).
- FIG 6
pH1N1 NP is less sensitive than sH1N1 NP to TRIM22-mediated ubiquitination. (A) sH1N1 Flag-NP, pH1N1 Flag-NP, and quadruple mutant (pH1N1-K4) were cotransfected with His-Ubi under all conditions and TRIM22 in HEK293T cells. WCEs were prepared from a 10% fraction of the cells and analyzed by Western blotting using anti-Flag (upper panel), anti-TRIM22 (middle panel), and anti-β-actin (lower panel) antibodies. (B) The remaining 90% of the cells were lysed under denaturing conditions, and ubiquitinated proteins were precipitated using Ni-NTA agarose. Purified ubiquitinated proteins were analyzed by Western blotting for the presence of ubiquitinated forms of Flag-NP (Ubi-NP) using anti-Flag antibodies. (C) The Flag-NP pH1N1 WT and its different K1, K2, K3, and K4 mutants were coexpressed with His-Ubi and TRIM22, in HEK293T cells. WCEs were prepared and analyzed as described for panel A. (D) Ubiquitinated forms of Flag-NP and its mutants (Ubi-NP) were analyzed as described for panel B. MW, molecular weight in thousands.
- FIG 7
Four arginine-to-lysine residues render the sH1N1 NP sensitive to TRIM22 restriction in a viral growth assay. (A) Recombinant viruses were generated starting from a pandemic H1N1 system. These viruses were used to infect control and human TRIM22-transduced MDCK cells at an MOI of 0.001. Substitution of pH1N1 NP with sH1N1 NP WT increased the susceptibility to TRIM22 restriction activity, whereas mutant viruses generated through the introduction of the mutation cluster composed of 4 K-to-R changes (mutant R4) into sH1N1 resulted in a gain of TRIM22 resistance. Viral titers are expressed as the means ± SD from three experiments performed in duplicate. P values were determined using two-way ANOVA (****, P < 0.0001). (B) WCEs from control and TRIM22-overexpressing MDCK cells infected with the reverse-genetics viruses were analyzed by Western blotting assay, and viral NP expression levels were evaluated. TRIM22 expression was also examined, and β-actin was used as a normalizer. These results are representative of one out of three independent experiments.
