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Fig. S1. A and B. HEK293 cells were infected with DENV DF and DENV DHF clinical isolates and hSec3p expression level was assessed at the indicated timings by CBF assay using anti-hSec3p and anti-actin antibody. Reduced hSec3p levels are detected in DENV DF and DENV DHF-infected cells from as early as 4 h p.i and are sustained up to 24 h p.i.

C and D. The levels of hSec3p measured by CBF assay following infection with DENV NGC (New Guinea C), DENV DF and DENV DHF virus isolates (moi of 0.1 and 1) were compared. *P < 0.05 compared with uninfected cells. **P < 0.05 compared with DENVNGC-infected cells.

Fig. S2. HEK293 cells were infected with UV-inactivated WNV/DENV and the levels of hSec3p, C and E proteins were measured using CBF assay. Significantly higher level of virus proteins (both C and E) is observed in WNV/DENV-infected cells compared with that of UV-inactivated WNV/DENV-infected cells at 4 h post infection. The hSec3p levels are significantly (P < 0.05) decreased in WNV-/DENV-infected cells compared with that of un-infected or UV-inactivated WNV/DENV. There is no significant difference (P > 0.05) in endogenous hSec3p levels between un-infected and UV-inactivated WNV/DENV-infected HEK293 cells. *P < 0.05 compared with the levels of virus protein in WNV/DENV-infected cells. **P < 0.05 compared with the levels of virus protein in un-infected cells.

Fig. S3. A and B. HEK293 cells were infected with (A) WNV or (B) DENV. The levels of hSec3p mRNA was measured using real-time PCR. There is no significant difference in the levels of hSec3p mRNA in WNV/DENV-infected cells compared with that of uninfected cells.

C and D. HEK293 cells were treated with actinomycin D and infected with (C) WNV or (D) DENV. The hSec3p expression levels were assessed at the indicated timings by CBF assay using anti-hSec3p and anti-actin antibody. The hSec3p levels are normalized against the actin control and the relative levels of hSec3p in virus-infected cells are shown. Reduced hSec3p level is detected in WNV/DENV-infected cells from as early as 4 h p.i. even in the presence of actinomycin D.

E and F. Effect of flavivirus infection on hSec6p expression following actinomycin D treatment. HEK293 cells were treated with actinomycin D and infected with (E) WNV or (F) DENV. The hSec6p expression levels were assessed at the indicated timings by CBF assay using anti-hSec6p and anti-actin antibody. There are no significant differences (P > 0.05) in the levels of hSec6p in virus-infected or uninfected cells.

G. Endogenous levels of hSec3p and hSec6p were measured in the presence of MG132, a proteasomal inhibitor. The levels of hSec3p and hSec6p are not altered significantly when MG132 is used. This confirmed that Sec3p and Sec6p levels were not controlled by proteasome in un-infected cells.

Fig. S4. HEK293 cells were transfected with WMycC and DMycC in the absence and presence of MG132. The expression of hSec6p is monitored by CBF assay using anti-hSec6p and anti-actin antibodies. The hSec6p levels are normalized against the actin control and the relative levels of hSec6p in transfected cells are shown.

Fig. S5. Interaction between WNV/DENV C protein and hSec3p. HEK293 cells were transfected with WMycC (WNV), DMycC (DENV) and hSec3p-binding defective mutants WMycC5′Δ15 (WNV)/DMycC5′Δ15 (DENV) plasmids. Cell lysates were immunoprecipitated with anti-hSec3p Ab and immunoblotted with anti-C Ab. The presence of bands in lanes 3 and 4 confirm C protein-hSec3p binding in full-length WNV/DENV C-transfected cells respectively.

Fig. S6. A–C. Proteolytic activities of 20S proteasome following transfection with WNV/DENV C protein in hSec3pKD cells. hSec3pKD cells were transfected with full-length C protein (WMycC/DMycC) or hSec3p-binding defective C mutants (WMycC5′Δ15/DMycC5′Δ15) and the proteolytic activities of 20S proteasome was examined. (A) Chymotrypsin-like and (C) caspase-like functions of 20S proteasome are slightly increased following transfection with full-length C protein of WNV/DENV. (B) No significant difference in the trypsin-like activity of 20S proteasome is observed following transfection with WNV/DENV C proteins in hSec3pKD cells.

D. Interaction between WNV/DENV C protein and hSec3p. HEK293 cells were transfected with WMycC (WNV), DMycC (DENV) and hSec3p-binding defective mutants WMycC5′Δ15 (WNV)/DMycC5′Δ15 (DENV) plasmids. Cell lysates were immunoprecipitated with anti-hSec3p Ab and immunoblotted with anti-C Ab. The presence of bands in lanes 3 and 4 confirm C protein–hSec3p binding in full-length WNV/DENV C-transfected cells respectively.

Fig. S7. Drug inhibition studies. HEK293 cells were treated with various inhibitors and chymotrypsin-like activity was measured. Full-length C protein of WNV/DENV activates the chymotrypsin-like activity in the presence of all the inhibitors except lactacystin.

Fig. S8. Multiple sequence alignment of WNV/DENV C proteins derived using CLUSTALW software (http://www.ebi.ac.uk/Tools/clustalw2/index.html). The accession numbers of WNV_S and DENV2 are AY688948 and FN429895 respectively.

Fig. S9. HEK293 cells were infected with viruses derived from infectious clones or infected with WNV and DENV (NGC/DF/DHF) in the presence of MG132 and lactacystin. The hSec3p levels and C protein levels were measured using CBF assay.

Table S1. Description of the plasmids encoding full-length WNV and DENV C protein.

Table S2. Description of the plasmids encoding hSec3p-binding defective WNV and DENV C protein.

Table S3. Description of the plasmids encoding various mutants of WNV and DENV C protein.

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