Figure 1.
Reovirus mutant jin-1 is able to infect the JAM-A negative cell line U118MG.
(A) Viability assay (WST-1) on 911 and U118MG cells. Cells were mock infected (black bar) or infected with wt T3D (white bar) or jin-1 virus (grey bar) with an MOI of 10, six days post infection. Means (± standard deviation) from three wells. (B) Detection of outer capsid protein σ3 in 911 and U118MG cells after addition of wt T3D or jin-1 virus with an MOI of 5. 40 hr post infection cells were stained with a monoclonal antibody directed against σ3 (4F2) and visualised with a fluorescein isothiocyanate (FITC)-conjugated goat-anti-mouse secondary antibody. The nuclei are visualised with 4′,6-diamidino-2-phenylindole (DAPI). (C) Assessment of reoviral protein synthesis in jin-1 or wt T3D infected cells. Indicated cells were infected with wt T3D or jin-1 virus and labeled with [35S]-methionine once CPE became apparent. 911 cells were infected with an MOI of 1 and U118MG or U118HAJAM cells with MOI of 5. Indicated are the positions of the reoviral λ, µ and σ proteins. m represents mock infected cells; wt: wt T3D infected cells and jin-1: jin-1 infected cells.
Table 1.
Amino acid differences in reovirus proteins of the jin mutants and the wt T3D reovirus strain.
Figure 2.
Schematic representation of mutations in Sigma-1.
Model of the σ1 protein (adapted from Chappel et al, 2009). Arrows indicate the positions of the mutations.
Figure 3.
Comparison of wt T3D with jin-viruses in terms of plaque size, yield in U118MG cells and primary human fibroblasts (VH10 cells).
(A) Yield of wt T3D and jin-1, jin-2 and jin-3 mutants from U118MG cells infected with MOI 10 per virus. Yields were determined 72 hours post infection by plaque assays on 911 cells. The graph shows yields (Log10 PFU) of two independent U118MG cell infections: first one is shown as a white bar and second as a grey bar. The dashed line represents the input amount of the initial infection. (B) Yield of wt T3D and jin-1, jin-2 and jin-3 mutants from VH10 cells infected with MOI 10 per virus. Yields were determined 72 hours post infection by plaque assays on 911 cells. The graph shows yields (Log10 PFU) of two independent VH10 cell infections: first one is shown as a white bar and second as a grey bar. The dashed line represents the input amount of the initial infection. (C) Plaque sizes of wt T3D and jin-1, jin-2 and jin-3 mutants in 911 cells. Surface areas of 10 plaques per virus were measured four days post infection with Olympus DP-software.
Figure 4.
Effect of the cysteine protease inhibitor E64d on jin-1, wt T3D virus, and ISVP entry into cells.
(A) U118MG cells were exposed to purified wt T3D virus and wt T3D ISVP (2*103 particles per cell). Lysates were made 24 hours post-infection and analyzed by SDS-PAGE and western-blotting. The reovirus σ3 proteins were detected by the anti-reovirus σ3 antibody 4F2 and an anti-Actin serum was used to detect actin as a loading control. (B) Effect of 100 µM E64d on the entry of particles compared to entry of ISVPs. Cells, treated (+) or untreated (−) with 100 µM E64d, were exposed to jin-1 (U118MG and 911 cells) or wt T3D (911 cells) virus or ISVPs (2*103 particles per cell). Lysates were made 24 hours post-infection. Equal amounts of protein were loaded on 10% SDS-polyacrylamide gel and detected with anti-reovirus σ3 antibody (4F2), and anti-Actin as a loading control.
Figure 5.
Analysis of Sigma-1 trimers synthesized in vitro.
(A) Top view of σ1-trimer, with colored monomer units (green, turquoise, orange). Position of the Q336R mutation in each monomer is indicated as red CPK symbol (Chappell et al., 2002); PDB ID: 1KKE. The software used for the 3D graphs is Viewerlite 5.0 from Accelrys. (B) [35S] methionine labelled in vitro transcribed and translated products of plasmids pDGC-S1wt (S1wt), pDGC-S1Q336R (S1Q336R) and pDGC-S1Y313A (S1Y313A) were incubated for 30 minutes at 37°C (to stabilize the mature trimers) or boiled for 5 minutes (to disrupt the trimers), before loading on a 10% SDS-polyacrylamide gel at 4°C. The position of the three different conformations is indicated.
Figure 6.
S1-sequence analysis of the jin-1/wt T3D and jin-2/wt T3D selection assay on U118 MG cells.
The jin-1 or jin-2 viruses were mixed with a 100-fold excess of wt T3D virus with regards to MOI. 911 cells were exposed to the mixtures first, before propagation on U118MG cells for three more passages. Reovirus RNA was isolated from the virus derived from the third passage on U118MG cells and subjected to RT-PCR to obtain the S1 products from the total population (jin-1/wt end or jin-2/wt end). Sequence histograms of the indicated regions were compared to the S1 sequences of the input reoviruses. Arrows indicate the nucleotide differences between the wt T3D and jin-1 or jin-2. (S1 nucleotide positions 571, 590 and 1019).
Figure 7.
Reovirus mutant jin-1 can infect cell lines that resist wt T3D reovirus infection.
(A) Several cell lines were infected with wt T3D or jin-1 and 32 hr post-infection cells were lysed. Protein samples (30 µg) were analyzed by 10% SDS-polyacrylamide gel electrophoresis. For the immunodetection anti-reovirus σ3 (4F2) was used. The cell lines 911 and U118-HAJAM are included to serve as positive controls for the infectivity of wt T3D. The cells were mock-infected (m); wt T3D infected, or jin-1 infected. (B) Virus production of wt T3D and jin-1 in the different cell lines. Cells were exposed to virus at MOI of 10 for one hour, washed with PBS and immediately lysed (1 hour time point) or left for 48 or 72 hours. For 911 cells an additional harvest point at 32 hours post-infection was included. The viral titers in the samples were determined by plaque assays on 911 cells. The graph shows a representative example of the assay. Open circles: wt T3D(o), crosses: jin-1(x). The dashed line represents the input amount of the initial infection (10 PFU/cell).
Figure 8.
Lec2 cells are poorly infected by reovirus mutants jin-1 and jin-3.
(A) CMP-sialic acid transporter defective Lec2 cells and parental cell line, CHO, were exposed to wt T3D, jin-1, and jin-3 at MOI of 10. Protein lysates were made 32 hrs post-infection and analyzed by SDS-polyacrylamide gel electrophoresis. For the immunodetection of σ3 the anti-reovirus σ3 antserum 4F2 was used, and anti-actin (human) was used to detect actin as a loading control. (B) Virus production of wt T3D and jin-1 in CHO and Lec2 cells. Cells were exposed for one hour to the viruses at MOI of 10, washed with PBS and immediately lyzed (1 hour time point), or incubated at 37°C for 48 hrs and 72 hrs. Virus yields were determined by plaque assays on 911 cells. The graph shows a representative example of the assay. Open circles: wt T3D(o), crosses: jin-1(x). The dashed line represents input amount of virus used at the initial infection (10 pfu/cell).
Figure 9.
WGA inhibits binding of reovirus to cells.
(A) Detection of Sialic acids in cell lines (911, CHO, U118MG and Lec2) by FITC-labeled WGA immunofluorescence. (B) WGA inhibition of reovirus infection. Prior to exposure of reovirus jin-1, the cells (U118MG, CHO, and Lec2) were mock-treated (−) or treated with 100 µg/ml WGA for 1 hr. at 37°C (+). After exposure of the cells to the virus at 4°C the cells were washed with PBS and incubated for an additional 32 hours in a CO2 incubator before protein lysates were made. For the immunodetection of the σ3 protein the anti-reovirus σ3 (4F2) was used and anti-actin was used as a loading control. (C) WGA inhibition of wt T3D and jin-1 reovirus infection in 911 cells.