Article Figures & Data
Figures
- FIG 1
RSV NS2 increases ubiquitin expression. (A) 293T cells were transfected for 24 h with a constant amount of HA-ubiquitin and increasing (2-fold) amounts of HA-NS2 (from 6.25 ng to 400 ng). (B) 293T cells were transfected with HA-ubiquitin for 8 h before either mock infection or infection with recombinant virus rA2, rWT (rHA-NS2), ΔNS1, or ΔNS2 at an MOI of 3 for 16 h.
- FIG 2
RSV NS2 induces monoubiquitination. (A) Map of ubiquitin with seven lysine residues labeled. Ubiquitin mutant constructs contain one or more lysine residues mutated to arginine. (B) 293T cells were transfected for 24 h with wild-type HA-ubiquitin (Ub) or HA-ubiquitin mutants, with NS2 cotransfected where indicated.
- FIG 3
RSV NS2 truncations reduce NS2-induced ubiquitin expression. 293T cells were transfected for 24 h with HA-ubiquitin or cotransfected with a wild-type HA-NS2 or HA-NS2 truncation.
- FIG 4
RSV NS2's effect on ubiquitin expression is conserved in bovine RSV NS2. 293T cells were transfected for 24 h with HA-ubiquitin or cotransfected with HRSV subgroup A NS2, HRSV subgroup B NS2, bovine RSV NS2, or NS2 of pneumonia virus of mice (PVM).
- FIG 5
RSV NS2 ubiquitin mutants limit NS2-induced ubiquitination. (A) Adaptive-BLAST predictions for residues essential to HRSV and BRSV NS2-induced ubiquitination. Human, sheep, and bovine RSV NS2 amino acid sequences are shown, with predicted essential residues illustrated by red boxes (top 20 scoring residues). Black boxes indicate conserved human and bovine RSV NS2 residues selected for mutation to alanine (residues above the threshold are underlined). (B) 293T cells were transfected for 24 h with HA-ubiquitin or cotransfected with HA-NS2 WT or ubiquitin mutants. Data are representative of three experiments. (C) Densitometry analysis of the Western blot comparing ubiquitin expression to NS2 expression. Significance was calculated by Student's t test, with P values of ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), and ≤0.0001 (****). Ave, average; AA, amino acid.
- FIG 6
RSV NS2 ubiquitin combination mutants display an additive effect on NS2-induced ubiquitination. (A) 293T cells were transfected for 24 h with HA-ubiquitin or cotransfected with HA-NS2 WT or ubiquitin mutants. Data are representative of three experiments. (B) Densitometry analysis of the Western blot comparing ubiquitin expression to NS2 expression. Significance was calculated by Student's t test, with P values of ≤0.05 (*), ≤0.01 (**), ≤0.001 (***), and ≤0.0001 (****). TL, T36A L52A mutant.
- FIG 7
rRSV NS2 ubiquitin mutants affect host protein expression. (A) A549 cells were either mock infected or infected with the indicated rRSV HA-tagged NS2 viruses at an MOI of 3 for 22 h. (B) 293T cells were transfected with HA-ubiquitin for 8 h before mock infection or infection at an MOI of 3 for 16 h with the indicated rRSV. (C) Quantitative RT-PCR analysis with rRSV NS2 ubiquitin mutants.
- FIG 8
Viral growth is attenuated by RSV NS2 ubiquitin mutants in A549 and Vero cells. A549 (A) and Vero (B) cells were infected at an MOI of 0.01, and supernatant was harvested for 5 days postinfection. Mean viral titers (y axis) and standard errors of the means are shown for the indicated day postinfection (x axis).
Tables
- TABLE 2
Viral growth is attenuated by rRSV NS2 ubiquitin mutants in A549 and Vero cells
Virus Growth in A549 cellsa Growth in Vero cellsa Avg day 4 titer (log10 PFU/ml) P valueb Avg day 4 titer (log10 PFU/ml) P valueb rWT 5.130 5.549 rT36A 4.103 0.0031** 5.030 0.0012** rL52A 4.395 0.0011** 5.276 0.0612c rP92A 3.930 0.0019** 4.929 0.0093** rTL 3.756 0.0003*** 5.016 0.0160* rTP 3.728 0.0010** 4.961 0.0005*** rLP 3.728 0.0003*** 4.950 0.0005*** rTLP 3.593 <0.0001**** 4.672 <0.0001**** ΔNS2 3.409 <0.0001**** 5.024 0.0007*** ↵a Cells were infected at an MOI of 0.01, and supernatant was harvested daily for 5 days postinfection.
↵b P values were determined by Student's t test in comparison to results for the rWT virus. Levels of significance are indicated as follows: *, ≤0.05; **, ≤0.01; ***, ≤0.001; ****, ≤0.0001.
↵c Not significant.
