Fig 1.
Protein 169 is expressed early and localizes in cytoplasmic puncta.
(A) HeLa cells were either mock-infected or infected at 5 plaque-forming units (PFU)/cell with different VACV strains or cowpox virus. Cell lysates were prepared 16 h p.i., separated by SDS-PAGE and immunoblotted using antibodies against 169, VACV protein D8 or α-tubulin. (B) HeLa cells were infected with v169 at 10 PFU/cell in the presence or absence of AraC (40 μg/ml) for the indicated times. Cell lysates were resolved by SDS-PAGE and immunoblotted using the indicated antibodies. (C) HeLa cells were either mock-infected or infected with indicated viruses at 10 PFU/cell. After 7 h, cells were separated into nuclear and cytoplasmic fractions and analysed by SDS-PAGE and immunoblotting with the indicated antibodies. In all immunoblots, molecular size markers are indicated on the left in kDa. (D) HeLa cells were infected with v169 at 10 PFU/cell for the indicated times and at 2 PFU/cell for 16 h. Cells were fixed and stained with anti-169 purified antibody (green) and DAPI (blue). White lines are scale bars (20 μm).
Fig 2.
Protein 169 is present in cytoplasmic puncta and is excluded from factories.
(A) HeLa cells were infected with v169 at 10 PFU/per cell for 7 h before cells were fixed and stained with anti-169 purified antibody (green), DAPI (blue) and with antibodies against markers of different intracellular compartments (red): protein disulphide isomerase (PDI) for endoplasmic reticulum (ER), GM130 for Golgi apparatus (Golgi), clathrin for some membrane vesicles, transferrin for endosomes and protein S6 for 40S ribosomes. Mitochondria were stained by addition of mitotracker to live cells that were then fixed and stained with anti-169 and with DAPI. White lines are scale bars (10 μm). (B) Enlarged versions of the mitochondrial and 40S ribosome staining from panel A showing DAPI-stained nuclei and virus factories (blue) and protein 169 (green). White lines are scale bars (20 μm).
Fig 3.
Protein 169 does not affect virus replication or spread in vitro.
(A-C) BSC-1, TK-143 and RK-13 cells were infected with the indicated viruses at 50 PFU/well of a 6-well plate. After 72 h, cells were stained with crystal violet and the radius of plaques (n = 20 per virus) was measured on a Zeiss Axio Vert.A1 microscope with AxioCam MRc and Axiovision 4.8 software. Results are expressed as the mean of radii of plaque size ± SD. Data shown are from one representative experiment (n = 3). (D, E) BSC-1 cells were infected at 10 PFU/cell and the amount of cell-associated virus (D) and extracellular virus (E) was determined at different times p.i. by plaque assay. (F, G) As in (D, E) except that cells were infected at 0.05 PFU/cell. Results are expressed as the mean titer per sample ± SD log10 PFU. Data from one representative experiment (n = 2) are shown.
Fig 4.
Protein 169 inhibits protein expression after activation of several innate immune signaling pathways.
(A) HEK 293T cells were transfected in triplicate with an NF-κB reporter plasmid, TK-renilla luciferase and plasmids for expression of the indicated proteins. After 1 d the cells were stimulated with 75 ng/ml of TNF-α for 7 h or treated with the same medium lacking TNF-α. The luminescence of cell lysates was measured using a luminometer. These data are from one representative experiment (n = 3) and results are presented as the fold increase in luciferase expression. Firefly luciferase was normalized to renilla luciferase (internal control) and further normalized to the unstimulated samples ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. (B) Performed as in (A) but using an ISG56.1 reporter plasmid (responsive to IRF-3) and cells were transfected with poly I:C or Lipofectamine only. (C) Performed as in (A) but using an ISRE reporter plasmid. The cells were stimulated with 100 U/ml of IFN-α for 7 h. (D) HEK 293T cells were transfected with plasmids for expression of the indicated proteins in triplicate, and the following day the cells were mock-infected or infected with SeV for 24 h. CXCL10 in the supernatant was measured by ELISA. Data shown are from one representative experiment (n = 2) and results are expressed as concentration of CXCL10, estimated from a nonlinear standard curve, ± SD. Statistical comparison to GFP control used a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001, **** p < 0.0001. (E, F, G) A549 cells were transfected with plasmids for expression of the indicated proteins in triplicate and, after 24 h cells, were mock-stimulated or stimulated with 50 ng/ml of TNF-α for 7 h. Then mRNAs were extracted, cDNAs were prepared and RT-q-PCR was performed using ViiA 7 Real-Time PCR System (Life Technologies) using primers specific for IL-6 (E), NFκBia (F) and ICAM-1 (G). Data shown are from one representative experiment (n = 2) and results are expressed as cycle threshold (CT) values compared to HPRT levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, * p < 0.05, ** p <0.01.
Fig 5.
Protein 169 inhibits cap-dependent and FMDV IRES-dependent translation.
(A) HeLa cells were transfected in duplicate with plasmids encoding the indicated proteins or with empty vector (EV) control together with a plasmid expressing GFP.FLAG. After 16 h, EV-transfected cells were treated with cycloheximide (CHX) (10 μg/ml) for 7 h. Cell lysates were resolved by SDS-PAGE and immunoblotted using antibodies against α-tubulin, 169, rabbit anti-FLAG (FLAG (R)) and mouse anti-FLAG (FLAG (M)). The positions of molecular size markers in kDa are indicated on the left. (B) Performed as in (A) except that mRNAs were extracted from cells, cDNAs were synthesized and GFP mRNA levels were measured by RT-q-PCR. Results are expressed as CT values compared to HPRT mRNA levels ± SD. Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.001. Data shown are from one representative experiment (n = 3). (C, D) HEK 293T cells were transfected with a plasmid encoding a bicistronic RNA expressing firefly luciferase (FLuc, C) by a cap-dependent translation and renilla luciferase (RLuc, D) by foot and mouth disease virus (FMDV) IRES-dependent translation, together with plasmids for expression of the indicated proteins or EV control in quadruplicate. After 16 h the cells were treated with CHX (1 μg/ml) for 7 h. The relative amount of FLuc and RLuc was determined by luminescence and results are presented as the fold increase in luciferase expression normalized to the EV control ± SD. Data shown are one representative experiment (n = 4). (E, F) Performed as in (C), but in triplicate. mRNAs were extracted, cDNAs were prepared and mRNA levels of for 169 and RLuc were determined by RT-q-PCR. Results are expressed as CT values compared to GAPDH levels ± SD. Data shown are one representative experiment (n = 4). Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, ** p < 0.01, *** p < 0.01, **** p < 0.001. (G) Cell lysates from (A) were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies. A shorter exposure of FLAG is shown to improve visualization of N1.TAP. The positions of molecular size markers in kDa are indicated on the left.
Fig 6.
Puromycin labeling of nascent polypeptides.
(A, B) HEK 293 Trex 169 (A) or C6.TAP (B) cells were uninduced or induced with DOXY (1 μg/ml) for the indicated times. Nascent proteins were then labeled with 5 μg/ml of puromycin for 25 min. Cell lysates were normalized by BCA assay and were resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. Data shown are one representative experiment (n = 2). (C) HeLa cells were either mock-infected or infected with v169 and vΔ169 at 5 PFU/cell for the indicated times. The samples were processed as described for (A, B) above. Data shown are from one representative experiment (n = 2). The positions of molecular size markers in kDa are indicated on the left for all panels.
Fig 7.
Protein 169 increases 80S monosomes and decreases polysomes.
HEK 293 Trex 169 cells were uninduced (A) or induced (B) with DOXY (1 μg/ml) for 14 h. Cytoplasmic cells lysates were prepared and resolved on 10–50% sucrose density gradients (Methods). The gradient was fractionated and fractions monitored by absorbance (A254 nm). Indicated fractions were collected and proteins were extracted, resolved by SDS-PAGE and analyzed by immunoblotting using the indicated antibodies. The positions of molecular size markers in kDa are indicated on the left and right. WCL = whole cell lysate. Data shown are from one representative experiment (n = 4).
Fig 8.
Analysis of polysomes in cells expressing protein C6.
HEK 293 Trex C6.TAP cells were uninduced (A) or induced (B) with DOXY (1 μg/ml) for 14 h and then treated as described in Fig 7. Data shown are from one representative experiment (n = 3).
Fig 9.
Accumulated 80S monosomes do not dissociate in high salt.
(A, B) The experiment was performed as described in Fig 7 with HEK 293 Trex 169 cells induced with DOXY (1 μg/ml for 16 h). The cytoplasmic fraction was prepared in normal salt (100 mM KCl, A) or high salt (400 mM KCl, B) and analyzed on sucrose density gradients containing either 100 mM KCl (A) or 400 mM KCl (B). Data shown are from one representative experiment (n = 2). (C, D) HEK 293 Trex 169 cells were uninduced and the cytoplasmic fraction was prepared in normal salt (100 mM KCl, C) or high salt (400 mM KCl, D) and analyzed on sucrose density gradients containing either 100 mM KCl (C) or 400 mM KCl (D). Data shown are from one representative experiment (n = 2).
Fig 10.
Protein 169 inhibits CrPV IRES-dependent translation.
(A, B) HEK 293T cells were transfected with a plasmid encoding a bicistronic RNA expressing renilla luciferase (RLuc, A) by a cap-dependent translation and firefly luciferase (FLuc, B) by a CrPV IRES-dependent translation, together with plasmids for expression of A49.TAP, 169 or EV control in quintuplicate. After 7 h the cells were treated with CHX (1 μg/ml) for 16 h. The relative amount of RLuc and FLuc was determined by luminescence and results are presented as the fold increase in luciferase expression normalized to the EV control ± SD. Data shown are from one representative experiment (n = 5). Statistical analysis was performed using a two-tailed Student’s t-test with Welch’s correction where necessary, *** p < 0.01, **** p < 0.001. (C) Cell lysates from (A) were resolved by SDS-PAGE followed by immunoblotting with the indicated antibodies.
Fig 11.
Protein 169 affects VACV virulence in the intranasal model of infection.
(A) BALB/c mice (n = 5) were infected i.n. with 5 × 103 PFU of the indicated purified viruses and the weight change and signs of illness were monitored daily (Methods). (B, C) BALB/c mice (n = 5) were mock treated or infected i.n. with 5 × 103 PFU of the purified viruses and 24 h p.i. the mice were killed and cytokines (B) and chemokines (C) in BAL fluids were assessed by ELISA. (D, E, F) BALB/c mice were infected as in (A) and at the indicated times groups of mice (n = 5) were killed. Then the weights of the lungs were determined (D), BAL fluids were collected and the total number of cells in these fluids were counted (E), and the viral titers in lungs were determined by plaque assay (F). Data presented in (A-F) are from one representative experiment (n = 2) and results are expressed as the average ± SEM. Statistical analysis in all panels was performed using a two-tailed Student’s t-test, * p ≤ 0.05, ** p ≤ 0.01.
Fig 12.
Infection with vΔ169 induces enhanced recruitment of macrophages and T-cells.
(A) BALB/c mice (n = 5) were infected i.n. with 5 × 103 PFU of the purified viruses and at the indicated times groups of mice (n = 5) were sacrificed and cells in BAL fluids were recovered, counted, and stained to determine the absolute number of macrophages (A), neutrophils (B), lymphocytes (C), NK cells (D), B cells (E), T cells (F), CD4+ T cells (G), CD8+ T cells (H) from mock-infected and VACV-infected mice by flow cytometry. Data shown are from one representative experiment (n = 2) and results are expressed as average ± SEM. Statistical analysis in all panels was performed using a two-tailed Student’s t-test, * p < 0.05, ** p<0.01.
Fig 13.
vΔ169 generates better protection after challenge.
(A) BALB/c mice (n = 5) were infected i.n. with 5 × 103 PFU of the purified viruses and at day 28 the mice were challenged i.n. with 5 × 106 PFU of wild type virus and the weight change was determined daily. (B) BALB/c mice were infected as in (A) and sera were collected 28 days p.i. and assayed for neutralization of intracellular mature virus of VACV strain WR. The median value for each population is represented by a horizontal black bar. (C, D) BALB/c mice were infected as in (A) and at 28 days p.i. splenic lymphocytes were harvested and their ability to lyse uninfected YAC-1 (C) or VACV-infected P815 (D) cells was determined by chromium release assay. Data are presented as the percentage cell lysis at various effector to target (E:T) cell ratios. Data shown are from one representative experiment (n = 2) and results are expressed as the average ± SEM. Statistical analysis for all panels was performed using a two-tailed Student’s t-test, * p < 0.05.
Fig 14.
Protein 169 affects VACV virulence and immunogenicity in the intradermal model of infection.
(A) C57BL/6 mice (n = 5) were infected i.d. with 1 × 104 PFU in the ear pinna with purified viruses and the lesion sizes were monitored daily (Methods). (B) C57BL/6 mice were infected as in (A) and at the indicated times group of mice (n = 5) were killed and the viral titers in ears were determined by plaque assay. (C) C57BL/6 mice (n = 5) were infected i.d. with 169 recombinant viruses and after 28 days the mice were challenged i.n. with 5 × 106 PFU of v169 and the weight change was determined daily. Data shown are from one representative experiment (n = 2) and results are presented as the average ± SEM. Statistical analysis for all panels was performed using a two-tailed Student’s t-test, * p < 0.05.