Physical fitness components associated with performance in a multiple-sprint test.

PURPOSE: The 5-m repeat-sprint test (5-m RST) measures resistance to fatigue after repeated bouts of short-duration, high-intensity activity. This study determined the components of fitness associated with performance in 5-m RSTs. METHODS: Speed (10-m and 40-m sprints), strength (bench press), agility, strength endurance (pull-ups and push-ups), and aerobic power (20-m shuttle-run test) were measured in male provincial- or national-level rugby (n = 110), hockey (n = 59), and soccer (n = 55) players. RESULTS: Subjects with either high (HI) or low (LO) resistance to fatigue in the 5-m RST differed in body mass (76.9 +/- 11.6 kg vs 102.1 +/- 18.9 kg, HI vs LO, respectively, P < .001), agility (14.55 +/- 0.41 seconds vs 15.56 +/- 0.30 seconds, P < .001), bench press (86 +/- 20 kg vs 114 +/- 33 kg, P = .03), pull-ups (13 +/- 4 vs 8 +/- 5, P = .02), push-ups (56 +/- 12 vs 39 +/- 13, P = .002), and 20-m shuttle-run test (20-m SRT; 133 +/- 11 vs 87 +/- 12 shuttles, P < .001). Body mass, strength, and aerobic power were the best predictors of 5-m RST performance: 5-m RST = -1.274(mass) + 0.756(1RM bench press) + 2.053(number of 20-m SRT shuttles) + 549.409 (R2 = .66). CONCLUSIONS: Performance in the 5-m RST is predicted best by a combination of factors including body mass, strength, and aerobic ability, rather than by any single component of fitness

DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection.

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses

DDX3 contributed to IFN-I suppression and viral replication/transcription after arenavirus infection in human cells. A.

<p>DDX3 ko-1, DDX3 ko-2 and WT A549 cells were infected with LCMV Cl13 (M.O.I. 0.5) for the indicated times. Total RNA in cell lysates was extracted and normalized interferon transcript levels (<i>ifnb/gapdh)</i> determined by qRT-PCR and represented as relative fold expression. <b>B.</b> DDX3 ko-1 and WT A549 cells were transduced with empty-RV (EV-RV) or RV encoding DDX3 (DDX3-RV) before infection, and processed as in <b>A</b> for quantification of <i>ifnb/gapdh</i> transcripts via qRT-PCR (<b>B,</b> left panel) or determination of bioactive IFN-I levels in cell culture supernatants at indicated h.p.i. (<b>B,</b> right panel). <b>C.</b> DDX3 ko-1 and WT A549 cells were pre-incubated for 2 h and infected with LCMV Cl13 (M.O.I. 0.5) in the presence of anti-IFNAR mAb (IFNAR Ab, solid lines) or Isotype control (Iso Ab, broken lines), which were left for the remaining of the culture. Viral titers were determined at indicated h.p.i. <b>D</b>. Translation assay performed in HEK-293T cells treated with DDX3 or scrambled (Scr) siRNA. <b>E.</b> Minireplicon assay performed in DDX3 ko-1 or WT A549 cells. <b>F</b> and <b>G.</b> DDX3 ko-1 or WT cells were transfected with 0.4 μg of empty plasmid, plasmid expressing DDX3 (<b>F-G</b>) or the indicated point-mutant DDX3 (<b>G</b>) and used for minireplicon assay. 100% value was given to WT A549 transfected with empty plasmid. Data are representative of 2 (<b>A-C</b> & <b>E-F</b>) or 3 (<b>D & G</b>) independent experiments. * p<0.05, ** p<0.01, ***p<0.001. Stars represent: DDX3ko vs WT (blue) or DDX3 ko at the indicated h.p.i. versus DDX3 ko at time = 0 (red) (<b>A</b>), DDX3 ko-EV vs WT-EV (black) or vs DDX3 ko-RV-DDX3 (red) (<b>B</b>), DDX3 ko-IFNAR vs WT-IFNAR (black) or vs DDX3 ko-Isotype (red) (<b>C</b>).</p

DDX3 interacted with LASV and LCMV NPs and promoted LCMV growth in human cells.

<p><b>A.</b> A549 cells were transfected for 60h with targeting siRNAs specific for DDX3 or scrambled siRNA controls followed by infection with LCMV Cl13 (M.O.I. 0.005). Viral titers in cell culture supernatants harvested 24 and 72 h.p.i. are shown. <b>B.</b> A549 cells were transfected for 24h with plasmids encoding LCMV-NP-HA, LASV-NP-HA or HA-USP14, or infected with 3rLCMV-HA-GFP, lysed and immunoprecipitated with anti-HA agarose beads (IP HA); eluates were analyzed by Immunoblot (IB). Immunoblots with anti-DDX3 and anti-GAPDH (load control) Abs were performed in input samples. <b>C.</b> LCMV infected A549 cells (M.O.I 1) were stained for DDX3 (green), LCMV NP (red) and DAPI and processed for Confocal Microscopy. Graphs on the right represent overlapping NP and DDX3 fluorescence intensities. <b>D.</b> Schematics of the genome of rLCMV-NP-HA. White: ORFs of viral proteins. Pink: HA tag. Black: viral untranslated regions. <b>E.</b> A549 cells were infected with rLCMV-NP-HA or 3rLCMV-HA-GFP for 24h, lysed in buffer containing RNAseA 0.1 mg/ml, immunoprecipitated (IP) and analyzed by Immunoblot (IB). (>) indicates NP-HA cleaved band. <b>F.</b> Viral titers in supernatants from DDX3 ko-1, DDX3 ko-2, WT-pCas9 (control) and WT A549 cells infected with LCMV Cl13 (M.O.I. 0.5) were quantified at 24, 48 and 72 h.p.i. <b>G.</b> DDX3 ko-1 and WT A549 cells transduced with RV expressing DDX3 or empty-RV were infected with LCMV Cl13 (M.O.I. 0.5) for 24h and viral RNA levels (<i>lcmvgp)</i> were determined relative to <i>gapdh</i> by RT-qPCR and represented as relative fold expression. <b>(B</b>&<b>E)</b> Numbers on the right: MW (kDa). Data are representative of 2 (<b>A, E</b> and <b>G</b>) or 4 (<b>B, C</b> and <b>F</b>) independent experiments. * p<0.05, ** p<0.01, ***p<0.001. Stars colors represent: DDX3 vs. Scr1 (blue) or Scr2 (black) (<b>A</b>) and WT A549 vs DDX3 ko-1 (red) or vs DDX3 ko-2 (black) (<b>F</b>).</p

DDX3 interacted with JUNV NP and promoted JUNV growth in human cells.

<p><b>A.</b> A549 cells were transfected with plasmid encoding JUNV NP-HA for 24h, lysed and immunoprecipitated with anti-HA agarose beads (IP HA). Eluates (upper panel) or input samples (load control, lower panel) were analyzed by Immunoblotting (IB) with anti-DDX3 or anti-GAPDH, respectively. <b>B-I.</b> DDX3 ko-1, DDX3 ko-2 and WT A549 cells were infected with JUNV Candid 1 (<b>B-F</b>) or Romero (<b>G-I</b>) strains at the indicated M.O.I. for 24h. Cells were stained with anti-GP antibodies and Hoechst, for confocal microscopy. Representative images for infected cells are shown (<b>D</b>). Numbers of infected cells were determined by high-content quantitative image-based analysis (<b>B</b>-<b>C</b> and <b>G and I</b>). Normalized viral RNA levels (<i>junv/gapdh</i>) represented as relative fold expression (<b>E</b> and <b>F</b>) and absolute copy numbers (<b>H</b>) of viral RNA in tissue culture supernatants were determined by qRT-PCR. When indicated, (<b>C, F and I</b>) DDX3 ko and WT A549 cells were transduced with empty-RV (EV-RV) or RV encoding DDX3 (DDX3-RV) before infection, and processed for confocal microscopy <b>(C and I)</b> or qRT-PCR <b>(F)</b>. Data are representative of 2 independent experiments. * p<0.05, **p<0.01,*** p<0.005, ****p<0.001. Star colors represent: DDX3 ko-EV vs WT-EV (black) or vs DDX3 ko-RV-DDX3 (red) (<b>C</b>), WT vs DDX3 ko-1 (red) or DDX3 ko-2 (black) (<b>E</b> & <b>G</b>) and DDX3 ko-1 + EV-RV vs DDX3 ko-1 + DDX3-RV (red) or DDX3 ko-2 + EV-RV vs DDX3 ko-2 + DDX3-RV (black) (<b>I</b>).</p