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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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E2 interacts with KLC2 and is necessary and sufficient to mediate F12 interaction with KLC2.(A) SDS-PAGE and immunoblot analysis of KLC (αFlag-IP), F12 (αHA-IP) and E2 (αV5-IP) immunoprecipitations carried out in parallel on cell lysates generated from HEK TRex-F12-HAco expressing Flag-KLC (isoform 1 or 2 as indicated), and expressing F12-HA (induced by addition of doxycycline) and/or V5-E2 (by transfection of pcDNA3-V5-E2co) as indicated. Clarified cell lysates (Input) and immunoprecipitated samples were immunoblotted with αFlag, αHA, αV5, αKif5B and α-αtubulin. (B) The E2 immunoprecipitation (αV5-IP) was repeated in the presence of virus infection to confirm the KLC isoform specificity. In addition to ectopic expression of Flag-KLC, F12-HA and V5-E2 cells were infected at 5 PFU/cell with either vΔF12 or vΔE2 (as indicated) such that samples only expressed either F12 or E2 or both proteins. Clarified cell lysate samples (Input) were immunoblotted for VACV protein D8 (αD8) to control for equal infection levels. The positions of molecular mass standards (kDa) are shown on the left. (C) Model for the order of interaction of F12, E2 and KLC.
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ppat.1004723.g005: E2 interacts with KLC2 and is necessary and sufficient to mediate F12 interaction with KLC2.(A) SDS-PAGE and immunoblot analysis of KLC (αFlag-IP), F12 (αHA-IP) and E2 (αV5-IP) immunoprecipitations carried out in parallel on cell lysates generated from HEK TRex-F12-HAco expressing Flag-KLC (isoform 1 or 2 as indicated), and expressing F12-HA (induced by addition of doxycycline) and/or V5-E2 (by transfection of pcDNA3-V5-E2co) as indicated. Clarified cell lysates (Input) and immunoprecipitated samples were immunoblotted with αFlag, αHA, αV5, αKif5B and α-αtubulin. (B) The E2 immunoprecipitation (αV5-IP) was repeated in the presence of virus infection to confirm the KLC isoform specificity. In addition to ectopic expression of Flag-KLC, F12-HA and V5-E2 cells were infected at 5 PFU/cell with either vΔF12 or vΔE2 (as indicated) such that samples only expressed either F12 or E2 or both proteins. Clarified cell lysate samples (Input) were immunoblotted for VACV protein D8 (αD8) to control for equal infection levels. The positions of molecular mass standards (kDa) are shown on the left. (C) Model for the order of interaction of F12, E2 and KLC.

Mentions: To test if E2 was the only VACV protein required for F12 to associate with KLC2, plasmids expressing epitope-tagged codon-optimised E2 (E2co) carrying either an N-terminal V5-tag or HA-tag, driven by a human cytomegalovirus promoter were constructed (see Materials and Methods). HEK TRex-F12-HAco cells transfected with plasmids expressing Flag-KLC1 or 2 were co-transfected with the V5-E2co expressing plasmid or empty vector control in the absence of virus infection (Fig. 5A). F12-HA expression was induced by treating with doxycycline. Lysates were immunoprecipitated with α-Flag, α-HA and α-V5. Pull-down of Flag-KLC2 (Fig. 5A i) co-precipitated F12-HA only when E2 was present (Fig. 5A ii). However, V5-E2 co-precipitated with Flag-KLC2 whether or not F12 was present (Fig. 5A iii), indicating that F12 is not required for E2 to interact with the kinesin-1 complex. The published F12/E2 interaction [46] was confirmed by the observation that F12 co-precipitated with E2 (Fig. 5A ii) and this interaction was maintained in the reciprocal IP (Fig. 5A iii). The levels of F12-E2 co-precipitation did not differ when KLC1 or 2 was over-expressed. KLC1 did not co-precipitate with F12 to detectable levels in reciprocal IPs (Fig. 5A i and Fig. 5A ii) but some co-precipitation was observed for KLC1 and E2 (Fig. 5A i and Fig. 5A iii), although levels were much lower than observed with KLC2. Repeating the V5-E2 IP in infected cells (Fig. 5B) confirmed the observations using uninfected cells, except that the E2/KLC interaction showed a higher specificity for KLC2 in infected cells (Fig. 5B ii). Taken together, these data show that E2 interacts with KLC2 and F12 interacts with E2 to form the F12/E2/KLC2 complex (Fig. 5C).


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)

E2 interacts with KLC2 and is necessary and sufficient to mediate F12 interaction with KLC2.(A) SDS-PAGE and immunoblot analysis of KLC (αFlag-IP), F12 (αHA-IP) and E2 (αV5-IP) immunoprecipitations carried out in parallel on cell lysates generated from HEK TRex-F12-HAco expressing Flag-KLC (isoform 1 or 2 as indicated), and expressing F12-HA (induced by addition of doxycycline) and/or V5-E2 (by transfection of pcDNA3-V5-E2co) as indicated. Clarified cell lysates (Input) and immunoprecipitated samples were immunoblotted with αFlag, αHA, αV5, αKif5B and α-αtubulin. (B) The E2 immunoprecipitation (αV5-IP) was repeated in the presence of virus infection to confirm the KLC isoform specificity. In addition to ectopic expression of Flag-KLC, F12-HA and V5-E2 cells were infected at 5 PFU/cell with either vΔF12 or vΔE2 (as indicated) such that samples only expressed either F12 or E2 or both proteins. Clarified cell lysate samples (Input) were immunoblotted for VACV protein D8 (αD8) to control for equal infection levels. The positions of molecular mass standards (kDa) are shown on the left. (C) Model for the order of interaction of F12, E2 and KLC.
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ppat.1004723.g005: E2 interacts with KLC2 and is necessary and sufficient to mediate F12 interaction with KLC2.(A) SDS-PAGE and immunoblot analysis of KLC (αFlag-IP), F12 (αHA-IP) and E2 (αV5-IP) immunoprecipitations carried out in parallel on cell lysates generated from HEK TRex-F12-HAco expressing Flag-KLC (isoform 1 or 2 as indicated), and expressing F12-HA (induced by addition of doxycycline) and/or V5-E2 (by transfection of pcDNA3-V5-E2co) as indicated. Clarified cell lysates (Input) and immunoprecipitated samples were immunoblotted with αFlag, αHA, αV5, αKif5B and α-αtubulin. (B) The E2 immunoprecipitation (αV5-IP) was repeated in the presence of virus infection to confirm the KLC isoform specificity. In addition to ectopic expression of Flag-KLC, F12-HA and V5-E2 cells were infected at 5 PFU/cell with either vΔF12 or vΔE2 (as indicated) such that samples only expressed either F12 or E2 or both proteins. Clarified cell lysate samples (Input) were immunoblotted for VACV protein D8 (αD8) to control for equal infection levels. The positions of molecular mass standards (kDa) are shown on the left. (C) Model for the order of interaction of F12, E2 and KLC.
Mentions: To test if E2 was the only VACV protein required for F12 to associate with KLC2, plasmids expressing epitope-tagged codon-optimised E2 (E2co) carrying either an N-terminal V5-tag or HA-tag, driven by a human cytomegalovirus promoter were constructed (see Materials and Methods). HEK TRex-F12-HAco cells transfected with plasmids expressing Flag-KLC1 or 2 were co-transfected with the V5-E2co expressing plasmid or empty vector control in the absence of virus infection (Fig. 5A). F12-HA expression was induced by treating with doxycycline. Lysates were immunoprecipitated with α-Flag, α-HA and α-V5. Pull-down of Flag-KLC2 (Fig. 5A i) co-precipitated F12-HA only when E2 was present (Fig. 5A ii). However, V5-E2 co-precipitated with Flag-KLC2 whether or not F12 was present (Fig. 5A iii), indicating that F12 is not required for E2 to interact with the kinesin-1 complex. The published F12/E2 interaction [46] was confirmed by the observation that F12 co-precipitated with E2 (Fig. 5A ii) and this interaction was maintained in the reciprocal IP (Fig. 5A iii). The levels of F12-E2 co-precipitation did not differ when KLC1 or 2 was over-expressed. KLC1 did not co-precipitate with F12 to detectable levels in reciprocal IPs (Fig. 5A i and Fig. 5A ii) but some co-precipitation was observed for KLC1 and E2 (Fig. 5A i and Fig. 5A iii), although levels were much lower than observed with KLC2. Repeating the V5-E2 IP in infected cells (Fig. 5B) confirmed the observations using uninfected cells, except that the E2/KLC interaction showed a higher specificity for KLC2 in infected cells (Fig. 5B ii). Taken together, these data show that E2 interacts with KLC2 and F12 interacts with E2 to form the F12/E2/KLC2 complex (Fig. 5C).

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