Results

Flavivirus replicase proteins are present behind a membrane barrier and are required in catalytic amounts

As reported by us previously for JEV (Uchil & Satchidanandam, 2003b), trypsin treatment of heavy membrane fractions from WNV and DENV-infected PS cells did not compromise in vitro RdRp activity (Fig. 1, lanes 2-3 for WNV and 15-16 for DENV). This resistance to trypsin indicated the presence of a membrane barrier behind which not only the template RF as shown by us previously (Uchil & Satchidanandam 2003a) but also the protein components of the flaviviral replication machinery were located. Complete solubilization of this barrier with the ionic detergent DOC that was known to preserve RdRp activity is expected to render the replicase proteins NS5 and NS3 accessible to trypsin. In keeping with this premise, pretreatment with 1% DOC followed by trypsin digestion resulted in complete destruction of RdRP activity (Fig. 1, lanes 5-6, 11-12 and 18-19 for WNV, JEV and DENV, respectively). Analysis of the viral protein profile from metabolically labelled WNV, DENV and JEV-infected cells following trypsin digestion in the absence and presence of DOC showed that trypsin degraded most of the detectable viral replicase proteins even in intact membranes in the absence

Trypsin Treated Cells

16KP

RdRp assay

FIG. 1. Flaviviral replication complexes are present behind a membrane barrier. Heavy membrane (16KP) fractions from WNV (lanes 1-6), JEV (lanes 7-12) and DENV (lanes 14-19) infected cells were subjected to increasing concentrations of trypsin with (+) or without (—) prior treatment with 1% sodium deoxycholate (DOC) as depicted in the flow chart before carrying out the RdRp assays using [a32P] GTP. The position and identity of the three viral RNA species RI, RF and vRNA following partially denaturing 7 M urea 3% polyacrylamide gel electrophoresis (urea-PAGE) are denoted by the arrowheads.

16KP

No / DOC treatment

Trypsin treatment

Inactivate trypsin

RdRp assay

15 16

FIG. 1. Flaviviral replication complexes are present behind a membrane barrier. Heavy membrane (16KP) fractions from WNV (lanes 1-6), JEV (lanes 7-12) and DENV (lanes 14-19) infected cells were subjected to increasing concentrations of trypsin with (+) or without (—) prior treatment with 1% sodium deoxycholate (DOC) as depicted in the flow chart before carrying out the RdRp assays using [a32P] GTP. The position and identity of the three viral RNA species RI, RF and vRNA following partially denaturing 7 M urea 3% polyacrylamide gel electrophoresis (urea-PAGE) are denoted by the arrowheads.

of DOC without attendant loss of RdRp activity (Fig. 2). Thus trace amounts of NS5 and NS3 that are protected from trypsin digestion obviously suffice for manifesting the total detectable RdRp activity as previously demonstrated for JEV (Uchil & Satchidanandam 2003b).

Fractionation ofproteins from TX100 treated membrane-bound flaviviral replication complexes

Our earlier studies had revealed that the RNA in the flaviviral RC was housed within virus-induced double-membrane structures, where the inner vesicles harboured the double-stranded RF template used by the flaviviral replicase (Uchil & Satchidanandam 2003a). When we carried out trypsin digestion of TX100 treated 16KP fractions, as seen for samples not exposed to detergent, most of the detectable viral replicase proteins were degraded by this treatment (Fig. 2). JEV RdRp activity was sensitive to TX100 (Fig. 3A, lane 6), in contrast to RdRp activity of WNV which was reduced but not completely lost in the presence of this detergent (Fig. 3A, compare lanes 1 and 2), similar to that seen with KUN RdRp (Chu & Westaway 1987). This residual activity partitioned almost equally between the

P16 membranes

P16 membranes

Detergent treatment

Trypsin treatment

Assay with 32P-UTP

FIG. 2. Effect of in vitro trypsin treatment on metabolically labelled flaviviral proteins. 16KP fractions metabolically labelled with [35S]methionine-cysteine from WNV (lanes 1-4), DENV (lanes 5-8) and JEV (lanes 9-12) infected cells were subjected to trypsin at 4 °C for 20 min without (lanes 2, 6 and 10) or with prior treatment with 1% sodium deoxycholate (DOC; lanes 4, 8 and 12) or 1% triton X-100 (TX100; lanes 3, 7 and 11). The processed samples were elec-trophoresed on SDS-10% polyacrylamide gel followed by autoradiography. The dots indicate the locations of flavivirus proteins.

Detergent treatment

Trypsin treatment

Assay with 32P-UTP

FIG. 2. Effect of in vitro trypsin treatment on metabolically labelled flaviviral proteins. 16KP fractions metabolically labelled with [35S]methionine-cysteine from WNV (lanes 1-4), DENV (lanes 5-8) and JEV (lanes 9-12) infected cells were subjected to trypsin at 4 °C for 20 min without (lanes 2, 6 and 10) or with prior treatment with 1% sodium deoxycholate (DOC; lanes 4, 8 and 12) or 1% triton X-100 (TX100; lanes 3, 7 and 11). The processed samples were elec-trophoresed on SDS-10% polyacrylamide gel followed by autoradiography. The dots indicate the locations of flavivirus proteins.

pellet and supernatant fractions following centrifugation at 16 000 X g for 10 min (Fig. 3A, lanes 3 and 4).

The differential effect of TX100 on the RdRp activity of JEV prompted us to probe the identity of protein(s) that might be released from their association with the flavivirus replicase by this detergent. Metabolically labelled flaviviral proteins from infected cells were analyzed following detergent extraction. As previously demonstrated (Uchil & Satchidanandam 2003a), these individual vesicles could be sedimented in an ultracentrifuge at 150 000 X g for 5 h. Comparison of the proteins present in the pellet and supernatant fractions following ultracentrifugation using the high-resolution Tris-tricine buffer revealed a uniform enrichment of NS5 and NS3 of JEV in the pellet and NS1 in the supernatant fraction (Fig. 3B). Interestingly NS1' which is unique to JEV and has not been reported in the other two viruses was also quantitatively released by TX100 into the supernatant (Fig. 3B, lane 2).

Virus

WNV

TX100

JEV NT

FIG. 3. Effect of TX100 on in vitro flaviviral RdRp activity. (A) 16KP fractions from WNV (lanes 1-4), or JEV (lanes 5-6) were treated (T; lanes 2, 6) or not treated (N; lanes 1, 5) with 1% TX100 and the supernatant fraction was sedimented at 16 000 X g. In vitro RdRp assay was carried out and labelled RNA products were analyzed using urea-PAGE. (B) Metabolically labelled proteins from JEV-infected 16KP fractions extracted with 1% TX100 and sedimented at 16 000 X g to obtain pellet and supernatant fractions. Pellet (UP; pellet) and supernantant (US; supernatant) fractions obtained by ultra centrifugation at 150 000 X g of the latter were analysed on SDS-Tricine-10% PAGE system and the proteins visualized by autoradiography. The dots represent flavivirus-specific proteins absent in mock-infected cells with their putative identities mentioned alongside. The asterisks denote proteins of probable host origin.

UP US

IFT

.5 • 3

m

•E •1

• C/2a/4a? •2b

FIG. 3. Effect of TX100 on in vitro flaviviral RdRp activity. (A) 16KP fractions from WNV (lanes 1-4), or JEV (lanes 5-6) were treated (T; lanes 2, 6) or not treated (N; lanes 1, 5) with 1% TX100 and the supernatant fraction was sedimented at 16 000 X g. In vitro RdRp assay was carried out and labelled RNA products were analyzed using urea-PAGE. (B) Metabolically labelled proteins from JEV-infected 16KP fractions extracted with 1% TX100 and sedimented at 16 000 X g to obtain pellet and supernatant fractions. Pellet (UP; pellet) and supernantant (US; supernatant) fractions obtained by ultra centrifugation at 150 000 X g of the latter were analysed on SDS-Tricine-10% PAGE system and the proteins visualized by autoradiography. The dots represent flavivirus-specific proteins absent in mock-infected cells with their putative identities mentioned alongside. The asterisks denote proteins of probable host origin.

TX100 but not DOC leads to dissociation of NS1 ' protein of JEV from NS3 and NS5

The above observation pointed to a possible role for NS1' in the TX100-induced loss of JEV RdRp activity. Having further confirmed the identity of the released proteins as NS1 and NS1' by use of a high-resolution tris-tricine gel system (Fig. 4A) and by Western blotting to NS1 specific serum (Fig. 4B), we attempted to assess the interaction of NS1 and NS1' with NS5 and NS3 proteins in the replicase complex by radioimmunoprecipitation of detergent extracts from JEV-infected cells using anti-NS3 serum. As seen in Fig. 4C (lanes 4 and 6), a comparable amount of NS5 was coprecipitated from both DOC and TX100-extracted membrane fractions. In corroboration with the near complete dissociation of NS1 from NS5 and NS3 following ultracentrifugation (Fig. 4, A and B), insignificant quantities of NS1 was associated with NS3 and NS5 (Fig. 4C, lanes 4 and 6). In keeping

TX100

TX100

a NS3

-

-

-

+

-

+

Pre-immune

-

-

+

-

+

-

1% DOC

-

S

+

+

-

-

1% TX100

-

-

-

-

+

FIG. 4. NS1' is co precipitated along with NS5 and NS3 by mouse anti-NS3 serum. (A) [35S]methionine-cysteine labelled proteins from JEV-infected 16KP fractions were treated with 1% TX100 (T), fractionated into pellet (P) and supernatant (S) fractions following centrifuga-tion at 16 000 X g. and analysed on SDS-Tricine-10% PAGE and visualized by autoradiography. (B) Western blotting of 1% deoxycholate or 1% TX100 fractionated proteins as in (A) from JEV-infected 16KP fractions using rabbit anti-NS1 antibodies. (C) Radioimmunoprecipitation (RIP) analysis of proteins released from JEV-infected 16KP fractions using anti-JEV NS3 antibodies. Labelled proteins released into supernatant fractions after DOC (S; lane 2) or TX100 treatment were immunoprecipitated using mouse pre-immune serum (lanes 3 and 5) or mouse anti-JEV NS3 antibodies (lanes 4 and 6). Arrows indicate the NS1' and NS1 co precipitated with NS3 in DOC extracts of 16KP fractions. The dots represent flavivirus-specific proteins with their putative identities mentioned alongside. The asterisks represent proteins presumably of host origin.

with the ability of DOC-solubilized membranes to retain RdRp activity, multiple viral proteins were coprecipitated by anti-NS3 antibody from these extracts in contrast to TX100-extracted membranes (Fig. 4C, lanes 4 and 6). The most striking observation was however the sizeable quantities of NS1' that coprecipitated only from DOC but not TX100 treated JEV-infected 16KP fractions, suggesting a vital role for this alternatively processed form of NS1 in JEV RNA synthesis. This protein brought down by anti-NS3 antibodies was confirmed to be NS1' and not the similar sized envelope by Western blotting to antisera specific to the two proteins (data not shown).

NS1'

Control

vRNA^

NS1'

Control

Solubilize pellet fraction with 1% DOC

Incubate with NS1' 4 °C/ 30min

FIG. 5. Restoration of release function ofJEV RdRp activity using recombinant NS1' protein. (A) SDS-PAGE analysis of recombinant NS1' ofJEV expressed in E. coli. E. coli cells containing non-recombinant (C; lane 1) or recombinant pRSET B carrying the JEV NS1' gene (T, lane 2) were induced with IPTG, proteins in the cell lysates were analysed using SDS-10% PAGE and visualized by Coomassie blue staining. The 55 kDa recombinant NS1' protein was purified by Ni-NTA agarose affinity chromatography, (P; lane 3). (B) 16KP fractions were processed as depicted in the flowchart and RdRp assay with [a32P] GTP carried out at the end of the incubation period with increasing amount of recombinant NS1' (lanes 3—4) or 6 ng of control protein (lane 5). The labelled RNA products were analysed as before. Lanes 6 and 1 depict RdRp activity in 16KP fractions before (S) and after (T) TX100 extraction. Arrowheads denote the positions of RI, vRNA and RF.

16KP i

1% TX100 Treatment

Solubilize pellet fraction with 1% DOC

Incubate with NS1' 4 °C/ 30min

Assay

FIG. 5. Restoration of release function ofJEV RdRp activity using recombinant NS1' protein. (A) SDS-PAGE analysis of recombinant NS1' ofJEV expressed in E. coli. E. coli cells containing non-recombinant (C; lane 1) or recombinant pRSET B carrying the JEV NS1' gene (T, lane 2) were induced with IPTG, proteins in the cell lysates were analysed using SDS-10% PAGE and visualized by Coomassie blue staining. The 55 kDa recombinant NS1' protein was purified by Ni-NTA agarose affinity chromatography, (P; lane 3). (B) 16KP fractions were processed as depicted in the flowchart and RdRp assay with [a32P] GTP carried out at the end of the incubation period with increasing amount of recombinant NS1' (lanes 3—4) or 6 ng of control protein (lane 5). The labelled RNA products were analysed as before. Lanes 6 and 1 depict RdRp activity in 16KP fractions before (S) and after (T) TX100 extraction. Arrowheads denote the positions of RI, vRNA and RF.

Addition of exogenous NS1' restores release function of TX100 treated JEV replicase

In order to directly ascertain the contribution of NS1' to JEV RdRp activity, we depleted 16KP membranes obtained from JEV-infected cells of the endogenous NS1' by two rounds of extraction using 1% TX100 after which no RdRp activity was observed (Fig. 5B, lane 1). The residual membranes were completely solubi-lized using 1% DOC following which incorporation of label into RI species alone was observed (Fig. 5B, lane 2) denoting restoration of elongation step of RNA synthesis by this detergent. Addition of recombinant NS1' purified from E. coli expressing this protein (Fig. 5A) brought about a dramatic restoration of release function of RdRp activity (Fig. 5B, lanes 3 and 4) with significant amounts of label incorporated into the RF species indicating proficient generation of RF from rep-licative intermediates (RI) following release of vRNA. The lack of complete restoration of RdRp activity under these conditions however suggested that proteins other than NS1' either of viral or host origin, required by the replicase complex may have also been depleted by TX100 treatment.

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