SUPPLEMENTAL INFORMATION
Materials and Methods
Synthesis and purification of [35S]-methionine labeled reovirus for binding experiments
Reovirus was propagated in L929 cells in the presence of 100 μCi/mL [ 35S] methionine in
methionine-free DMEM supplemented with 5% dialyzed FBS (Invitrogen) (1). Subsequent [35S]methionine labeled reovirus was purified by lysing infected cells in 1% Igepal, 0.5% Sodium
deoxycholate, 50mM Tris pH 7.4, 150mM NaCl, 1x protease inhibitor cocktails (RIPA). Nuclear
and cellular debris were removed by twice centrifugation at 14 000 g for 10 minutes. Resulting
supernatant was applied to 30% sucrose cushion and subjected to high speed centrifugation at
100 000 g for 1h. Reovirus pellet was resuspended in PBS, and infectivity quantified via
standard plaque assay on L-929 monolayers. The purified virus typically had a specific activity
of 4000 cpm/107 pfu.
For binding experiments, 10 [35S]-methionine labeled reovirus pfu/cell for 1 hr at 4oC as
described in the “Reovirus infection and titers” section of the manuscript. Subsequently cell
monolayers were washed 3-times with PBS. Post wash, cells were collected by scraping, washed
and lysed with RIPA and radioactivity quantified using a Wallac, Winspectral 1414 liquid
scintillation counter (Perkin Elmer Life Sciences, Boston MA).
Northern analysis of reovirus mRNA and dsRNA
RNA of reovirus infected NIH 3T3 (pBabepuro or pBabepuro RasV12) cells was harvested,
purified, reverse transcribed and visualized by northern blotting at various time post infection
exactly as described in the Quantitative RT PCR and northern analysis section portion of the
manuscript. Positive sense and negative sense probes generated against S2, S4, M1, M2, and L1
genome segments were generated as described in the manuscript. To detect (-) RNA (represents
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dsRNA), total RNA in glyoxal loading dye (Ambion) was heated to 65oC (denatures mRNA and
dsRNA), separated, transferred and probed with digoxigenin-labeled RNA probes that were
complementary, as described in the manuscript.
Detection of reovirus protein shift from soluble to insoluble (viral factories) cellular
fractions
At 18 hpi, cells were metabolically labeled with 100 μCi/mL EasyTag [35S] methionine in
methionine-free DMEM for 30 min and either washed and lysed, or following pulse labeling,
monolayers were washed and chased for various time intervals in DMEM containing 10% NCS
prior to lysis. To isolate cytoskeletal inclusion bodies, washed monolayers were incubated with
10mM Pipes (pH 6.8), 3mM MgCl2, 100mM KCl, 30mM sucrose, 1% triton X-100, 1mM
PMSF, and PIC and soluble proteins collected. The remaining cytoskeleton and associated
structures were solubilized with RIPA/0.1% SDS. Metabolically-reovirus proteins in the cell
lysates were detected and quantified as described in the “Metabolic radiolabeling and
immunoprecipitation” section of the manuscript.
Electron microscopy
At 24 hpi, cells were harvested, pelleted, and fixed for 2 hr at RT in 3% glutaraldehyde, 0.25M
sucrose, 50mM sodium cacodylate buffer, then post-fixed in 2% OsO4 in the same buffer, and
incubated overnight in 0.1% aqueous uranyl acetate. Cells were embedded in TAAB resin and
dehydrated in ethanol.
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FIGURE LEGENDS
Figure S1. Quantification of cellular growth rates of reovirus infected non-transformed (○)and
Ras-transformed (■) NIH 3T3 cells (error bars +/- SD, n = 3).
Figure S2. Binding of [35S]-methionine labeled reovirus for 1 h at 4oC to non-transformed and
Ras-transformed NIH 3T3 cells (error bar + SD, n=3).
Figure S3. Reovirus induced cytopathic effects are visualized in non-transformed and Ras
transformed cells at up to 48 hpi. Substantial cell rounding and detachment is seen in Ras
transformed but not non-transformed cells.
Figure S4. Northern blot analysis of accumulating reovirus transcripts in infected non- and Rastransformed cells. (a) L1, M1, or S2 mRNAs were detected at 0, 18, and 24 hours post infection
(hpi). RNA samples were heated at 50oC and hybridized to (-) sense probes to detect mRNAs.
One-half dilutions of RNA from Ras transformed cells collected at 48 hpi were used to generate
curves of band intensity versus initial RNA amount and thereby determine accurately the fold
increase of each RNA species in Ras-transformed over non-transformed cells (as indicated below
each time point). (b) Reovirus M2 and S4 mRNA fold increases in Ras-transformed over nontransformed cells (Fig. 2b) were calculated from these serial dilutions of samples when the signal
is saturated in Fig. 2b. (a) and (b): All samples were normalized to the 18S ribosomal subunit as
detected with ethidium bromide. The Reo lane is dsRNA isolated from purified reovirus and
denatured at 65C.
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Figure S5. Western blot detecting accumulating reovirus proteins in non- and Ras-transformed
cells (top), or anti-actin (bottom), at various hpi. Densitometry quantification of individual
reovirus protein bands and the average fold increase in Ras-transformed cells are indicated in
table below
Figure S6. Virus assembly is not enhanced in Ras-transformed cells.(a) Virus assembly was
assessed by monitoring the shift of newly synthesized, Triton X-soluble (S) viral proteins to
insoluble (I) “viral factories”. Infected cells were metabolically labeled with [ 35S]-methionine at
18 hpi for 30 min (pulse), chased for up to 6 h, and subject to immunoprecipitation with antireovirus antibody. (b) Viral factories and particles detected in sections of non- and Rastransformed cells 24 hpi by TEM.
Figure S7. Northern blot analysis of accumulating dsRNA reovirus segments in infected nonand Ras-transformed cells. L1, M1, or S2 dsRNA segment were detected at 0, 18, and 24 hours
post infection (hpi). RNA samples were heated at 65oC and hybridized to (+) sense probes to
detect dsRNAs. One-half dilutions of RNA from Ras transformed cells collected at 48 hpi were
used to generate curves of band intensity versus initial RNA amount and thereby determine
accurately the fold increase of each RNA species in Ras-transformed over non-transformed cells
(as indicated below each time point). All samples were normalized to the 18S ribosomal subunit
as detected with ethidium bromide. The Reo lane is dsRNA isolated from purified reovirus and
denatured at 65C.
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Figure S8. Standard curves (top) showing Ct values versus relative starting template amount
demonstrate the linearity of QPCR using primers specific for (+) sense RNA or GAPDH mRNA.
Dissociation curves (bottom) suggest that only single PCR products are amplified by
corresponding primer pair.
References
1. Duncan, R. (1996) The Low pH-Dependent Entry of Avian Reovirus Is Accompanied by
Two Specific Cleavages of the Major Outer Capsid Protein μ2C, Virology 219, 179189.
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