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Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

Carpentier DC, Gao WN, Ewles H, Morgan GW, Smith GL - PLoS Pathog. (2015)

Bottom Line: Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress.In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways.Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.

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Endogenous KLC2 co-immunoprecipitates with E2.(A) SDS-PAGE and immunoblot analysis of α-HA-IP from HEK 293T cells infected with either vF12-HA or vB14-HA at 5 PFU/cell and harvested 14 hpi. Clarified cell lysates (Input) and α-HA immunoprecipitated samples were immunoblotted with the antibodies indicated on the left of the figure. (B) SDS-PAGE and immunoblot analysis of α-HA immunoprecipitation from lysates generated from HEK 293T cells transfected with a plasmid encoding HA-tagged E2 or a control plasmid as indicated. Samples were probed for the precipitated E2 protein (i) and for co-precipitation of KLC using the 63–90 antibody (ii). The co-precipitating KLC isoform identity was confirmed by immunoblotting with antibodies specific for KLC1 (iii) and KLC2 (iv). Co-precipitation of the entire kinesin-1 complex with E2 was confirmed by immunoblotting with the α-Kif5B antibody (v).
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ppat.1004723.g006: Endogenous KLC2 co-immunoprecipitates with E2.(A) SDS-PAGE and immunoblot analysis of α-HA-IP from HEK 293T cells infected with either vF12-HA or vB14-HA at 5 PFU/cell and harvested 14 hpi. Clarified cell lysates (Input) and α-HA immunoprecipitated samples were immunoblotted with the antibodies indicated on the left of the figure. (B) SDS-PAGE and immunoblot analysis of α-HA immunoprecipitation from lysates generated from HEK 293T cells transfected with a plasmid encoding HA-tagged E2 or a control plasmid as indicated. Samples were probed for the precipitated E2 protein (i) and for co-precipitation of KLC using the 63–90 antibody (ii). The co-precipitating KLC isoform identity was confirmed by immunoblotting with antibodies specific for KLC1 (iii) and KLC2 (iv). Co-precipitation of the entire kinesin-1 complex with E2 was confirmed by immunoblotting with the α-Kif5B antibody (v).

Mentions: As mentioned, attempts to precipitate endogenous KLC with F12-HA were unsuccessful. This was also true using larger amounts of cell lysate, larger volumes of anti-HA antibody-coated beads and longer incubation times (Fig. 6A). However, because the results shown in Fig. 4 and Fig. 5 indicated that E2 can interact with KLC2 without F12, interactions between endogenous kinesin-1 (KLC and KHC) and epitope-tagged E2 were investigated. The HA-tagged E2co plasmid was transfected into 293T cells, E2co was precipitated with anti-HA beads and samples were analysed by immunoblotting using the 63–90 antibody. This detected several bands in the input lanes (Fig. 6B ii) corresponding to the different KLC isoforms present (possibly including variants of KLC1 produced by differential splicing [56]). Notably, a band corresponding to one of the larger isoforms was detected co-precipitating with HA-E2 but not in the negative control (Fig. 6B ii). In the literature this upper band is often assumed to correspond to KLC2 with the lower band corresponding to KLC1 [31,51]. To confirm that the co-precipitated protein was KLC2, immunoblotted membranes were stripped and re-probed with antibodies specific for KLC1 (Fig. 6B iii) and KLC2 (Fig. 6B iv). Only the anti-KLC2 antibody recognised the band co-precipitating with E2, although the entire kinesin-1 complex co-precipitated with E2 because KHC was also detected (Fig. 6B v). These results indicate that E2 interacts with endogenous human KLC2 but not KLC1 and this specificity is indistinguishable from the murine KLCs expressed ectopically. This retention of this specificity between the human and murine proteins is consistent with the very high amino acid conservation of these proteins (S1 Fig.).


Vaccinia virus protein complex F12/E2 interacts with kinesin light chain isoform 2 to engage the kinesin-1 motor complex.

Carpentier DC, Gao WN, Ewles H, Morgan GW, Smith GL - PLoS Pathog. (2015)

Endogenous KLC2 co-immunoprecipitates with E2.(A) SDS-PAGE and immunoblot analysis of α-HA-IP from HEK 293T cells infected with either vF12-HA or vB14-HA at 5 PFU/cell and harvested 14 hpi. Clarified cell lysates (Input) and α-HA immunoprecipitated samples were immunoblotted with the antibodies indicated on the left of the figure. (B) SDS-PAGE and immunoblot analysis of α-HA immunoprecipitation from lysates generated from HEK 293T cells transfected with a plasmid encoding HA-tagged E2 or a control plasmid as indicated. Samples were probed for the precipitated E2 protein (i) and for co-precipitation of KLC using the 63–90 antibody (ii). The co-precipitating KLC isoform identity was confirmed by immunoblotting with antibodies specific for KLC1 (iii) and KLC2 (iv). Co-precipitation of the entire kinesin-1 complex with E2 was confirmed by immunoblotting with the α-Kif5B antibody (v).
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ppat.1004723.g006: Endogenous KLC2 co-immunoprecipitates with E2.(A) SDS-PAGE and immunoblot analysis of α-HA-IP from HEK 293T cells infected with either vF12-HA or vB14-HA at 5 PFU/cell and harvested 14 hpi. Clarified cell lysates (Input) and α-HA immunoprecipitated samples were immunoblotted with the antibodies indicated on the left of the figure. (B) SDS-PAGE and immunoblot analysis of α-HA immunoprecipitation from lysates generated from HEK 293T cells transfected with a plasmid encoding HA-tagged E2 or a control plasmid as indicated. Samples were probed for the precipitated E2 protein (i) and for co-precipitation of KLC using the 63–90 antibody (ii). The co-precipitating KLC isoform identity was confirmed by immunoblotting with antibodies specific for KLC1 (iii) and KLC2 (iv). Co-precipitation of the entire kinesin-1 complex with E2 was confirmed by immunoblotting with the α-Kif5B antibody (v).
Mentions: As mentioned, attempts to precipitate endogenous KLC with F12-HA were unsuccessful. This was also true using larger amounts of cell lysate, larger volumes of anti-HA antibody-coated beads and longer incubation times (Fig. 6A). However, because the results shown in Fig. 4 and Fig. 5 indicated that E2 can interact with KLC2 without F12, interactions between endogenous kinesin-1 (KLC and KHC) and epitope-tagged E2 were investigated. The HA-tagged E2co plasmid was transfected into 293T cells, E2co was precipitated with anti-HA beads and samples were analysed by immunoblotting using the 63–90 antibody. This detected several bands in the input lanes (Fig. 6B ii) corresponding to the different KLC isoforms present (possibly including variants of KLC1 produced by differential splicing [56]). Notably, a band corresponding to one of the larger isoforms was detected co-precipitating with HA-E2 but not in the negative control (Fig. 6B ii). In the literature this upper band is often assumed to correspond to KLC2 with the lower band corresponding to KLC1 [31,51]. To confirm that the co-precipitated protein was KLC2, immunoblotted membranes were stripped and re-probed with antibodies specific for KLC1 (Fig. 6B iii) and KLC2 (Fig. 6B iv). Only the anti-KLC2 antibody recognised the band co-precipitating with E2, although the entire kinesin-1 complex co-precipitated with E2 because KHC was also detected (Fig. 6B v). These results indicate that E2 interacts with endogenous human KLC2 but not KLC1 and this specificity is indistinguishable from the murine KLCs expressed ectopically. This retention of this specificity between the human and murine proteins is consistent with the very high amino acid conservation of these proteins (S1 Fig.).

Bottom Line: Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress.In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways.Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of Cambridge, Cambridge, United Kingdom.

ABSTRACT
During vaccinia virus morphogenesis, intracellular mature virus (IMV) particles are wrapped by a double lipid bilayer to form triple enveloped virions called intracellular enveloped virus (IEV). IEV are then transported to the cell surface where the outer IEV membrane fuses with the cell membrane to expose a double enveloped virion outside the cell. The F12, E2 and A36 proteins are involved in transport of IEVs to the cell surface. Deletion of the F12L or E2L genes causes a severe inhibition of IEV transport and a tiny plaque size. Deletion of the A36R gene leads to a smaller reduction in plaque size and less severe inhibition of IEV egress. The A36 protein is present in the outer membrane of IEVs, and over-expressed fragments of this protein interact with kinesin light chain (KLC). However, no interaction of F12 or E2 with the kinesin complex has been reported hitherto. Here the F12/E2 complex is shown to associate with kinesin-1 through an interaction of E2 with the C-terminal tail of KLC isoform 2, which varies considerably between different KLC isoforms. siRNA-mediated knockdown of KLC isoform 1 increased IEV transport to the cell surface and virus plaque size, suggesting interaction with KLC isoform 1 is somehow inhibitory of IEV transport. In contrast, knockdown of KLC isoform 2 did not affect IEV egress or plaque formation, indicating redundancy in virion egress pathways. Lastly, the enhancement of plaque size resulting from loss of KLC isoform 1 was abrogated by removal of KLC isoforms 1 and 2 simultaneously. These observations suggest redundancy in the mechanisms used for IEV egress, with involvement of KLC isoforms 1 and 2, and provide evidence of interaction of F12/E2 complex with the kinesin-1 complex.

Show MeSH
Related in: MedlinePlus