Species-Specific Inhibition of RIG-I Ubiquitination and IFN Induction by the Influenza A Virus NS1 Protein

Influenza A viruses can adapt to new host species, leading to the emergence of novel pathogenic strains. There is evidence that highly pathogenic viruses encode for non-structural 1 (NS1) proteins that are more efficient in suppressing the host immune response. The NS1 protein inhibits type-I interferon (IFN) production partly by blocking the TRIM25 ubiquitin E3 ligase-mediated Lys63-linked ubiquitination of the viral RNA sensor RIG-I, required for its optimal downstream signaling. In order to understand possible mechanisms of viral adaptation and host tropism, we examined the ability of NS1 encoded by human (Cal04), avian (HK156), swine (SwTx98) and mouse-adapted (PR8) influenza viruses to interact with TRIM25 orthologues from mammalian and avian species. Using co-immunoprecipitation assays we show that human TRIM25 binds to all tested NS1 proteins, whereas the chicken TRIM25 ortholog binds preferentially to the NS1 from the avian virus. Strikingly, none of the NS1 proteins were able to bind mouse TRIM25. Since NS1 can inhibit IFN production in mouse, we tested the impact of TRIM25 and NS1 on RIG-I ubiquitination in mouse cells. While NS1 efficiently suppressed human TRIM25-dependent ubiquitination of RIG-I 2CARD, NS1 inhibited the ubiquitination of full-length mouse RIG-I in a mouse TRIM25-independent manner. Therefore, we tested if the ubiquitin E3 ligase Riplet, which has also been shown to ubiquitinate RIG-I, interacts with NS1. We found that NS1 binds mouse Riplet and inhibits its activity to induce IFN-β in murine cells. Furthermore, NS1 proteins of human but not swine or avian viruses were able to interact with human Riplet, thereby suppressing RIG-I ubiquitination. In conclusion, our results indicate that influenza NS1 protein targets TRIM25 and Riplet ubiquitin E3 ligases in a species-specific manner for the inhibition of RIG-I ubiquitination and antiviral IFN production

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Chimeric mouse TRIM25 containing the CCD of human TRIM25 recovers NS1 binding.

<p>(<b>A</b>) Schematic representations of human TRIM25 (hT25), mouse TRIM25 (mT25) and the mouse/human TRIM25 chimera (mChimT25_h191–379). Numbers indicate amino acid. (<b>B</b>) WCLs of HEK293T cells, that had been transfected with empty vector, V5-hTRIM25, V5-mTRIM25 or V5-mChimT25_h191–379 together with NS1-PR8, were subjected to IP with anti-V5 antibody, followed by IB with anti-NS1. (<b>C</b>) Localization of NS1-PR8 in HeLa cells determined by confocal microscopy. At 30 h posttransfection with NS1-PR8 alone, NS1-PR8 together with V5-hTRIM25, V5-mTRIM25, or V5-mChimT25_h191–379, HeLa cells were stained with anti-NS1 (green), anti-V5 (red) and DAPI (nucleus, blue). (<b>D</b>) Quantification of cytoplasmic NS1 localization from (C). From three independent experiments, 50 cells with expression of NS1 alone or NS1 together with hTRIM25, mTRIM25 or mChimT25_h191–379, respectively, were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test.</p

Influenza A Virus NS1 interacts with mouse Riplet.

<p>(<b>A</b>) At 30 h posttransfection with Myc-tagged mouse Riplet (Myc-mRiplet) together with NS1-PR8, HEK293T cells were mock-treated or infected with SeV (10 HA units/ml) for 10 h. V5-tagged hTRIM25 was co-transfected as positive control. WCLs were subjected to IP with anti-Myc (Riplet) or anti-V5 (TRIM25), followed by IB with anti-NS1 antibody. (<b>B</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies. (<b>C</b>) Localization of NS1-PR8 and mouse Riplet in HeLa cells determined by confocal microscopy. At 30 h posttransfection with Myc-mRiplet alone, NS1-PR8 alone, or NS1 together with Myc-mRiplet, HeLa cells were stained with anti-Myc (green), anti-NS1 (red), and DAPI (nucleus, blue). Fifty cells from three independent experiments were counted and the percentage of cells with cytoplasmic NS1 is shown, ***p<0.001 by Fisher's exact test. (<b>D and E</b>) Hepa1.6 cells were transfected with Flag-tagged mouse Riplet. At 30 h posttransfection, cells were either mock-treated or infected with recombinant A/PR/8/34 virus expressing NS1 PR8, Cal04, HK156, Tx91, SwTx98, or Pan99 at an MOI of 2 (<b>D</b>), or R38A/K41A or E96A/E97A NS1 mutant at an MOI of 4 (<b>E</b>). 18 h later, WCLs were subjected to IP with anti-NS1 antibody, followed by immunoblotting using the indicated antibodies.</p

NS1 proteins from human influenza strains bind and inhibit human Riplet.

<p>(<b>A</b>) HEK293T cells were transfected with empty vector or HA-tagged human Riplet (HA-hRiplet). At 30 h posttransfection, cells were either mock-treated, or infected with the indicated recombinant A/PR/8/34 viruses at an MOI of 2. 18 h later, WCLs were subjected to IP with anti-HA antibody, followed by immunoblotting using the indicated antibodies. (<b>B</b>) Tx91 recombinant virus suppresses the endogenous RIG-I ubiquitination more potently than PR8 virus. HEK293T cells, that had been transfected with HA-tagged ubiquitin, were either mock-treated, or infected with ΔNS1 PR8, PR8 WT, or Tx91-NS1 recombinant virus at an MOI of 2 for 18 h. WCLs were subjected to IP with anti-RIG-I antibody, followed by IB with anti-HA or anti-RIG-I antibody. Expression of HA-ubiquitin, viral NS1, and Actin was further determined in the WCLs. (<b>C</b>) A549 cells were infected with PR8-NS1 or Tx91-NS1 recombinant virus at an MOI of 0.1. Cells were collected at the indicated time points and IFN-β mRNA was measured by qPCR. (<b>D and E</b>) A549 cells were transiently transfected with non-silencing control siRNA (si.C), or with siRNA specific for TRIM25 (si.TRIM25), Riplet (si.Riplet), or both. At 40 h posttransfection, cells were infected with PR8 WT or Tx91 recombinant virus at an MOI of 2 for 30 h. The mRNA levels of TRIM25 and Riplet were measured by qPCR for analyzing their knockdown efficiency (<b>D</b>). Furthermore, IFN-β mRNA levels were assessed by qPCR (<b>E</b>). <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003059#s2" target="_blank">Results</a> are triplicates from 3 independent experiments. NS; statistically non-significant.</p

NS1 inhibits the Riplet-dependent RIG-I ubiquitination and IFN induction in murine cells.

<p>(<b>A</b>) Mouse Hepa 1.6 cells were transfected with vector or Flag-tagged mRiplet with or without NS1-PR8. WCLs were subjected to IP with RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. Expression of mRiplet, NS1, and ubiquitin (Ub) was determined in the WCLs by IB with anti-Flag, anti-NS1 or anti-Ub antibody. (<b>B</b>) Mouse Hepa1.6 cells were transfected with IFN-β luciferase reporter plasmid together with empty vector or mRIG-I together with or without mRiplet and NS1-PR8. At 24 h posttransfection, cells were lysed and subjected to luciferase assay. Data shown is representative of 3 independent experiments and depicted is the mean ± SD (n = 3). (<b>C</b>) Influenza NS1 protein specifically inhibits the Riplet-dependent ubiquitination of mouse RIG-I. Hepa1.6 cells were transiently transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet) together with empty vector or NS1-PR8. At 24 h posttransfection, cells were infected with SeV (50 HA units/ml) for 22 h. WCLs were used for IP with anti-RIG-I antibody, followed by IB with anti-Ub or anti-RIG-I antibody. (<b>D–F</b>) Knockdown of endogenous Riplet in mouse embryonic fibroblasts enhances influenza A virus replication. WT or <i>TRIM25 −/−</i> MEFs were transfected with non-silencing control siRNA (si.C), or with a siRNA specific for mouse Riplet (si.Riplet). At 30 h posttransfection, cells were infected with recombinant A/PR/8/34 WT virus (MOI 0.1). Knockdown of endogenous Riplet was confirmed by RT-PCR (<b>D</b>). Supernatants were assayed for progeny virus yields 24 h postinfection in standard plaque titrations (<b>E</b>). Virus yields are depicted in Pfu/ml. The results of three independent experiments are shown. Furthermore, viral NS1 protein expression was determined in the WCLs of infected cells (<b>F</b>).</p