Vaccinia virus A36R membrane protein provides a direct link between intracellular enveloped virions and the microtubule motor kinesin

Abstract

Previous work demonstrated that intracellular enveloped vaccinia virus virions use microtubules to move from the site of membrane wrapping to the cell periphery. The mechanism and direction of intracellular virion movement predicted that viral proteins directly or indirectly interact with the microtubule motor protein kinesin. The yeast two-hybrid assay was used to test for interactions between the light chain of kinesin and the cytoplasmic tails from five viral envelope proteins. We found that the N-terminal tetratricopeptide repeat region of the kinesin light chain (KLC-TPR) interacted with the cytoplasmic tail of the viral A36R protein. A series of C- and N-terminal truncations of A36R further defined a region from residues 81 to 111 that was sufficient for interaction with KLC-TPR. Interactions were confirmed by using pull-down assays with purified glutathione S-transferase (GST)-A36R and (35)S-labeled KLC-TPR. The defined region on A36R for interaction with kinesin overlaps the recently defined region (residues 91 to 111) for interaction with the A33R envelope protein. The yeast three-hybrid system was used to demonstrate that expression of A33R interrupted the interaction between A36R and KLC-TPR, indicating that the binding of A36R is mutually exclusive to either A33R or kinesin. Pull-down assays with purified GST-A36R and (35)S-labeled KLC-TPR in the presence of competing A33R corroborated these findings. Collectively, these results demonstrated that the viral A36R protein interacts directly with the microtubule motor protein kinesin and that the viral protein A33R may regulate this interaction.

FIG. 1.

Yeast two-hybrid assay. The cytoplasmic domains of various IEV proteins were fused to the GAL4 BD (BD-Fusion) and tested for interaction with KLC-TPR that had been fused to the GAL4 AD (AD-Fusion) by cotransfection of the two plasmids into yeast. DDO medium was streaked with cotransformed yeast cells to confirm the presence of both BD and AD fusions. Positive interactions were determined by growth on QDO medium.

FIG. 2.

In vitro interaction assay. Fusion proteins were overexpressed in E. coli and affinity purified with glutathione resin. (A) Purified proteins were analyzed by SDS-PAGE followed by Coomassie blue staining. (B) GST pull-down assays were carried out with either GST-A36R^24-111, GST, or buffer alone bound to glutathione resin and incubated with in vitro ³⁵S-labeled KLC-TPR. The masses (in kilodaltons) and positions of migration of markers are indicated on the left. Complexes were analyzed by SDS-PAGE. A PhosphorImager was used to detect and quantify the amount of radioactivity in each band with the relative amounts shown at the bottom.

FIG. 3.

In vivo interaction assay. GST pull-down assays were carried out with either GST-A36R^24-111 or GST bound to glutathione resin and incubated with a HeLa cell cytoplasmic extract. Complexes were analyzed by SDS-PAGE followed by Western blotting with either anti-KHC MAb (αKHC) or anti-KLC MAb (αKLC). The masses (in kilodaltons) and migration positions of markers are indicated on the left.

FIG. 4.

Yeast three-hybrid interaction assay. Yeast strain CG1945 was cotransfected with pBridgeBD-KLC-TPR/A36R^24-111 and pADA33R^1-40. (A) Extracts from cotransformed yeast that had been grown in medium with (+) or without (−) methionine were analyzed by SDS-PAGE followed by Western blotting with anti-HA MAb. The masses (in kilodaltons) and positions of migration of markers are indicated on the left. (B) Tenfold dilutions of overnight cultures of cotransformed yeast were applied as spots to selective medium with (+) or without (−) methionine and histidine.

FIG. 5.

Yeast three-hybrid competition assay. Yeast strain CG1945 was cotransfected with pBridgeBD-KLC-TPR/A33R^1-40 and pADA36R^24-111. (A) Extracts from cotransformed yeast that had been grown in medium with (+) or without (−) methionine were analyzed by SDS-PAGE followed by Western blotting with anti-HA MAb. The masses (in kilodaltons) and migration positions of markers are indicated on the left. (B) Tenfold dilutions of overnight cultures of cotransformed yeast were applied as spots to selective medium with (+) or without (−) methionine and histidine.